Biomarker-based methods for identifying and formulating compositions that improve skin quality and reduce the visible signs of aging in skin

ABSTRACT

In various embodiments, provided are methods for selecting and formulating compositions for treating and maintaining the quality of skin, wherein a composition is selected for use in a personal care product based on its demonstrated biological effect to improve skin quality as evidenced by one or more biomarker changes that correlate with improvement as evidenced by one or more objective measurements of skin health.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to PCT Application No. US2010/040074filed Jun. 25, 2010, which claims priority to the following: U.S.Provisional Application Ser. Nos. 61/295,732 filed Jan. 17, 2010, and61/295,826 filed Jan. 18, 2010, and PCT Applications Nos. US2010/022454,US2010/022456, US2010/022455, and US2010/022458 all filed Jan. 28, 2010,the disclosures of which are incorporated herein by reference in theirentirety. This application is related to U.S. application Ser. Nos.12/478,624 filed Jun. 4, 2009 and 12/361,492 filed Jan. 28, 2009, thedisclosures of which are incorporated herein by reference in theirentirety.

FIELD

The present disclosure relates to methods for identifying compositionsthat can improve the health of epithelial tissue. It also relates to theuse of such materials selected by such methods, for formulating andpreparing at least one personal care composition.

BACKGROUND

The signs of skin damage and aging include, but are not limited to,outward visibly and tactilely perceptible changes that include but arenot limited to: textural discontinuities such as wrinkles and coarsedeep wrinkles, fine lines, crevices, bumps, large pores, unevenness,roughness, sagging, puffiness; blotchiness and sallowness;hyperpigmentation, including discoloration, spots and freckles;keratoses, abnormal differentiation, hyperkeratinization, elastosis, andcollagen breakdown; and loss of one or more of elasticity, firmness,tightness, and recoil from deformation. Such signs may be caused orinduced by factors that are intrinsic to the aging body, or areextrinsic, such as environmental damage. Skin damage and aging involvesone or more layers of epithelial tissue, and is often most evident inthe stratum corneum, the outermost layer of skin, though damage andchanges may present in any of the layers of the epithelia/epidermis,dermis, underlying tissues and vasculature.

The consumer products and cosmetics industries are focused, at least inpart, on providing products to consumers that reduce the signs of skindamage and aging. Such products may be exclusively designed to improvethe appearance of skin, though many include one or more other functionalbenefits, such as cleansing, delivery of color, texture or scent, andexfoliation, to name a few. To date, little scientific data exists todemonstrate whether and how cosmetics and other products influence thequality or health of skin. Indeed, most products do little more thancoat and temporarily moisturize or soften the stratum corneum, and thesebenefits are transient and superficial. In recent years, basicscientific research and applied pharmaceutical research has expanded theunderstanding of the biochemical processes that underlie tissue damageand tissue aging, including skin. This developing scientific knowledgehas influenced both the cosmetics and personal care products industries,and its influence is evident in the contexts of cleansing and otherpersonal care products as well as cosmetics.

In recent years, consumers have been presented with a panoply ofproducts that purport to provide scientifically-based solutions to skinaging by providing actives with antioxidant, anti-inflammatory andfree-radical-scavenging effects, to name a few. Yet it remains the casewith many products that the actual cellular and physiological effects onconsumers, when measured against objective standards, are lacking,though the actives may exhibit the claimed properties in the laboratory.Moreover, there is a distinct lack of evidence regarding how and in whatdoses product actives, whether new or old, impact the quality and healthof skin. This lack of clinical understanding inherently limits effectiveformulation of products and delivery regimens that confer actualimprovement to skin tissues and cells.

The present inventors recognized the disconnects between the skinscience and the existing knowledge about skin care products.

SUMMARY

Accordingly, it is an objective of the present invention to apply thebasic science knowledge about biomarkers and skin health in a rigorousand objective manner to identify and evaluate test agents for usefulnessin personal care products to improve skin health. Methods are providedwhich enable identification and characterization of agents whichpositively influence cellular and tissue properties to maintain orrestore health to the skin. Representative compositions are describedwhich are tested and positively identified as influencing staticallysignificant changes in a variety of skin biomarkers. These biomarkerchanges are shown to correlate closely with objective measures ofimproved skin health. A representative panel of biomarkers includesinflammatory cytokines, natural moisturizing factors, keratin 1, keratin10, keratin 11, lipids and total protein.

These and other features, aspects, and advantages of the embodimentsdisclosed herein will become evident to those skilled in the art from areading of the present disclosure with the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows representative results with two different constant lipidproducts (commercially available) wherein the products provide varyingskin conditioning when delivered over a time period of 21 days, withmeasurements taken for dryness change at days 7, 14 and 21.

FIG. 2 shows an exemplary profile of lathering phase to hydrophobicbenefit phase.

FIG. 3 shows a delivery profile for an exemplary personal carecomposition comprising lathering phase and hydrophobic benefit phasewherein the ratio of lathering phase to hydrophobic benefit phase variesacross a treatment cycle from about 70:30 through about 45:55 to about80:20, wherein the volume of dispensed composition is 250 ml.

FIG. 4 shows results of cytokine biomarker measurement comparing waterand a test composition in a representative clinical study, demonstratingthat test compositions can affect measurable and significant changes inskin biology.

FIG. 5 shows the correlation of objective physical measurement oftrans-epidermal water loss with results of cytokine biomarkermeasurement in a representative clinical study, demonstrating thatbiomarker measurements correlate well with measurement of skin health.

FIG. 6 shows the correlation of objective physical measurement of skincohesiveness on dryness improvement with results of total proteinbiomarker measurement in a representative clinical study at 24 hours,demonstrating that biomarker measurements correlate well withmeasurement of skin health.

FIG. 7 shows the correlation of objective physical measurement of skincohesiveness on dryness improvement with results of total proteinbiomarker measurement in a representative clinical study at 48 hours,demonstrating that biomarker measurements correlate well withmeasurement of skin health.

FIG. 8 shows the correlation of objective physical measurement oftrans-epidermal water loss improvement with results of lipid (ceramides)biomarker measurement in a representative clinical study, demonstratingthat biomarker measurements correlate well with measurement of skinhealth.

FIG. 9 shows the lipid delivery profile according to an embodiment asdescribed herein wherein the ratio of lathering phase to hydrophobicbenefit phase varies across a treatment cycle from about 70:30 throughabout 45:55 to about 80:20.

FIG. 10 shows the relative change in visual dryness using water, apersonal care composition having a lathering phase to hydrophobicbenefit phase ratio of 55:45 and a personal care composition having thelipid delivery profile as shown in FIG. 9.

FIG. 11 shows the relative change in skin condition measured with acorneometer using water, a personal care composition having a latheringphase to hydrophobic benefit phase ratio of 55:45 and a personal carecomposition having the lipid delivery profile as shown in FIG. 9.

FIG. 12 shows the relative change in skin trans epidermal water lossusing water, a personal care composition having a lathering phase tohydrophobic benefit phase ratio of 55:45 and a personal care compositionhaving the lipid delivery profile as shown in FIG. 9.

FIG. 13 shows the change in skin deformation over time.

FIG. 14 shows the relative change in Ue using water, a personal carecomposition having a lathering phase to hydrophobic benefit phase ratioof 55:45 and a embodiment of personal care composition having the lipiddelivery profile as shown in FIG. 9.

FIG. 15 shows the relative change in Ur using water, a personal carecomposition having a lathering phase to hydrophobic benefit phase ratioof 55:45 and a personal care composition having the lipid deliveryprofile as shown in FIG. 9.

FIG. 16 shows the relative change in total protein using water, apersonal care composition having a lathering phase to hydrophobicbenefit phase ratio of 55:45 and a personal care composition having thelipid delivery profile as shown in FIG. 9.

FIG. 17 shows the relative change in keratin 1, 10 and 11 normalized tosoluble protein using water, a personal care composition having alathering phase to hydrophobic benefit phase ratio of 55:45 and apersonal care composition having the lipid delivery profile as shown inFIG. 9.

FIG. 18 shows plots of objective physical measurement of corneometerimprovement as a function of combined biomarker measurements.

FIG. 19 shows plots of objective physical measurement of trans-epidermalwater loss improvement as a function of combined biomarker measurements.

FIG. 20 shows plots of objective physical measurement of elasticrecovery improvement as a function of combined biomarker measurements.

FIG. 21A shows plots of predictability of skin dryness as a function ofcombined biomarkers including log IL 1ra/1α, NMF, and total proteinbiomarker measurements.

FIG. 21B shows plots of dryness improvement as a function ofinflammatory NMF.

FIG. 21C shows plots of dryness improvement as a function ofinflammatory cytokines (log IL 1ra/1α).

FIG. 21D shows plots of dryness improvement as a function of totalprotein.

DETAILED DESCRIPTION

The present invention is based in part of discovery by the inventorsthat beneficial effects of consumer personal care products can bedetected within the tissue and cells using one or more tissue andcellular biomarkers. The inventors identified and characterized a panelof biomarkers that demonstrate statistically significant changes withinskin tissue in response to treatment with test agents. In manyinstances, these changes have been demonstrated by the inventors toclosely correlate with objective measures of skin health. As a result ofthe inventors' efforts, the invention provides in some embodiments apanel of biomarkers, one or more of which can be used as an indicator ofpositive benefits of one or more test agents on skin. In some aspects,the invention also includes specific correlations between one or more ofthe biomarkers and one or more objective measures of skin health.Therefore, use of the biomarker panels enables the efficient screeningand identification of test agents as providing one or more beneficialeffects for use in any of a variety of formulations for consumer use.

It will be appreciated by one of skill in the art that a testcomposition which demonstrates beneficial effects based on biomarkermeasurement may be useful in cosmetic and other personal care products,including leave-on and rinse-off formulations. Since rinse-offcompositions are not left on the skin to provide continued delivery ofbenefit agents, it is an especially great challenge in the art toprovide rinse-off products that have beneficial effects that persistafter the product is rinsed away. As described herein in particulardetail in the examples, the screening methods are ideal for formulatingrinse-off products which are typically contacted with a consumer's skinfor only a brief time.

It is known that skin quality is not maintained during sporadictreatment with rinse-off compositions, generally. And it is known thatskin quality is not maintained or even improved using rinse-offcompositions that comprise relatively low hydrophobic benefit phase, forexample compositions comprising ratios of lathering phase to hydrophobicbenefit phase that are in the range from more than 90:10 to about 60:40(by weight). FIG. 1 shows representative results with two differentconstant lipid products (commercially available) wherein the productsprovide varying skin conditioning when delivered over a time period of21 days, with measurements taken for dryness change at days 7, 14 and21. Referring again to FIG. 1, only the 50% lipid shows a significantsustained benefit with continued use as compared with water. Incontrast, the 30% lipid shows only a modest improvement over time ascompared with water.

These results together with the results described in the Examples hereinbelow show that the measured improvement in skin quality conferredduring the treatment stage (the period of delivery of a hydrophobicbenefit phase in a rinse-off product) persists well beyond the cessationof treatment. These results were unexpected. Even more unexpected werethe results as described herein relating to the positive change invarious biomarkers associated with treatment with hydrophobic benefitphase and the correlation of those positive changes with measuredphysical properties of skin. Notably, these results are particularlysurprising since the tested skin was not classified as diseased.

Accordingly, developed in accordance with the methods described herein,the test formulations include compositions that are selected for theirdemonstrable ability to enhance skin health. Most moisturizing bodywashes are designed to address dryness using surface moisturizers, butthis is only a temporary fix for dry skin. Personal care compositionssuch as body washes, formulated in accordance with the inventive methodsprovide benefits beyond moisturizing skin at the surface anddemonstrably penetrate the skin to improve overall skin health at thecellular level, as evidenced by changes in molecular biomarkers thatcorrelate with objective measures of skin health. This is an advancementover the art, and surprisingly enables provision of specially formulatedcompositions and treatment regimens.

I. Definitions

“Biomarker” as used herein refers to any biological molecules (genes,proteins, lipids, metabolites) that, singularly or collectively, reflectthe current or predict future state of a biological system. Thus, asused herein, various biomarkers are indicators of the quality of skin interms of elasticity including elastic extension and elastic recovery,firmness, tissue hydration, brightness, tone, smoothness, appearance oflines, visual properties of dryness and condition, sagging, the presenceof flaking, cohesiveness as evidenced by total protein, lipid content,and trans-epidermal water loss. Non-limiting examples of biomarkersinclude inflammatory cytokines, natural moisturizing factors, one ormore of keratins 1, 10 and 11, lipids and total protein. The response ofskin to treatment with personal care compositions can also be assessedby measuring one or more biomarkers.

“Comprising” as used herein is inclusive and does not excludeadditional, unrecited elements, steps or methods. Terms as used hereinthat are synonymous with “comprising” include “including,” “containing,”and “characterized by,” and mean that other steps and other ingredientscan be included. The term “comprising” encompasses the terms “consistingor” and “consisting essentially of,” wherein these latter terms areexclusive and are limited in that additional, unrecited elements, stepsor methods ingredients may be excluded. The personal cleansingcompositions and methods of the present disclosure can comprise, consistof, or consist essentially of, the elements, steps and methods asdescribed herein.

“Consumer” as used herein refers to an individual who purchases and/oruses compositions in accordance with the disclosure. In some instances,therefore, a consumer may be alternately referred to herein as a “user.”

“Control surfaces” as used herein means a region of epithelial tissuewhich has not been contacted with and/or by the product, such as aconsumer product and/or a test product, which has contacted the affectedsurface. Typically, control surface is will be of similar epithelialtissue which has not contacted the product. Alternatively, the controlsurface may be the same epithelial tissue as the affected surface. Inthis case the control surface is harvested some time prior, such as from1 second to 5 or 6 weeks or possibly even longer to the application ofthe test product and/or comparison product to the epithelial tissue,which is then harvested to collect the test sample.

“Effective amount” as used herein means an amount of a compound orcomposition sufficient to significantly induce a positive skin benefit,including independently or in combination with other benefits disclosedherein. This means that the content and/or concentration of activecomponent in the formulation is sufficient that when the formulation isapplied with normal frequency and in a normal amount, the formulationcan result in the treatment of one or more undesired skin conditions(e.g., skin wrinkles). For instance, the amount can be an amountsufficient to inhibit or enhance some biochemical function occurringwithin the skin. This amount of active component may vary dependingupon, among other factors, the type of product and the type of skincondition to be addressed.

“Epidermis” as used herein refers to the outer layer of skin, and isdivided into five strata, which include the: stratum corneum, stratumgranulosum, stratum spinosum, and stratum basale. The stratum corneumcontains many layers of dead, anucleated keratinocytes that areessentially filled with keratin. The outermost layers of the stratumcorneum are constantly shed, even in healthy skin. The stratum lucidumcontains two to three layers of anucleated cells. The stratum granulosumcontains two to four layers of cells that are held together bydesmosomes that contain keratohyaline granules. The stratum spinosumcontains eight to ten layers of modestly active dividing cells that arealso held together by desmosomes. The stratum basale contains a singlelayer of columnar cells that actively divide by mitosis and provide thecells that are destined to migrate through the upper epidermal layers tothe stratum corneum. Thus, the predominant cell type of the epidermis isthe keratinocyte. These cells are formed in the basal layer and existthrough the epidermal strata to the granular layer at which theytransform into the cells know as corneocytes or squames that form thestratum corneum. During this transformation process, the nucleus isdigested, the cytoplasm disappears, the lipids are released into theintercellular space, keratin intermediate filaments aggregate to formmicrofibrils, and the cell membrane is replaced by a cell envelope madeof cross-linked protein with lipids covalently attached to its surface.Thus, keratins are the major structural proteins of the stratum corneum.Corneocytes regularly slough off (a process known as desquamation) tocomplete an overall process that takes about a month in healthy humanskin. In stratum corneum that is desquamating at its normal rate,corneocytes persist in the stratum corneum for approximately 2 weeksbefore being shed into the environment.

“Epithelial tissue” as used herein refers to all or any portion of theepithelia, in particular the epidermis, and includes one or moreportions of epithelia that may be obtained from a subject by aharvesting technique known in the art, including those described herein.By way of example and without being limiting, epithelial tissue refersto cellular fragments and debris, proteins, isolated cells from theepithelia including harvested and cultured cells. Some non-limitingexamples of sources of epithelial tissue include, skin on the face,head, torso and limbs of a subject.

“Hydrophobic benefit phase” as used herein, refers to the compositionthat comprises one or a combination of hydrophobic benefit materialsthat deliver one or more benefits including skin conditioning, skinmoisturization, and skin health benefits. The term “lipid” is usedherein in reference to hydrophobic benefit phases. In accordance withsome embodiments, hydrophobic benefit phases are selected from the groupconsisting of petrolatum, lanolin, derivatives of lanolin (non-limitingexamples include lanolin oil, isopropyl lanolate, acetylated lanolin,acetylated lanolin alcohols, lanolin alcohol linoleate, lanolin alcoholriconoleate) hydrocarbon oils (e.g. mineral oil) natural and syntheticwaxes (non-limiting examples include micro-crystalline waxes, paraffins,ozokerite, lanolin wax, lanolin alcohols, lanolin fatty acids,polyethylene, polybutene, polydecene, pentahydrosqualene) volatile ornon-volatile organosiloxanes and their derivatives (non-limitingexamples include dimethicones, cyclomethicones, alkyl siloxanes,polymethylsiloxanes, methylphenylpolysiloxanes), natural and synthetictriglycerides (non-limiting examples include castor oil, soy bean oil,sunflower seed oil, maleated soy bean oil, safflower oil, cotton seedoil, corn oil, walnut oil, peanut oil, olive oil, cod liver oil, almondoil, avocado oil, palm oil, sesame oil), and combinations thereof.

“Lathering phase” as used herein refers to the composition thatcomprises a surfactant, which when combined with water and mechanicallyagitated generates a foam or lather sufficient to cause a personal carecomposition to provide a lather, and which, when tested using the TotalLather Volume Method disclosed herein, yield lather volumes in the rangefrom 800 ml to more than 1500 ml.

“Package” includes any suitable container for personal carecompositions.

“Personal care article” as used herein, refers to a delivery means (suchas a “Package”) comprising a “personal care composition.”

“Personal care composition” as used herein, refers to compositionsintended for topical application to the epithelia, including skin andhair. The compositions used in accordance with the present disclosureinclude topically applied compositions, including leave-on formulations,and rinse-off formulations in which the product is applied topically tothe skin or hair and then is subsequently rinsed within minutes from theskin or hair with water, or otherwise wiped off using a substrate withdeposition of a portion of the composition. Such rinse off compositionsmay be used as body washes, moisturizing body wash, shampoo,conditioning shampoo, hair conditioner, shower gels, skin cleansers,cleansing milks, hair and body wash, in shower body moisturizer, petshampoo, shaving preparations and cleansing compositions used inconjunction with or applied to a disposable cleansing cloth and othertypes of products. The personal care composition used in accordance withthe present disclosure is typically dispensible from a package. Thus, insome embodiments, the dispensing may be by extruding. In someembodiments the package may be a single chamber package, or a multichamber package, or a set of discrete packages. The personal carecompositions used in accordance with the present disclosure can be inthe form of liquid, semi-liquid, cream, lotion or gel compositionsintended for topical application to skin.

“Premium experience stage” refers to stages in which the components in apersonal care composition are associated with delivery of one or moreexperiential benefits to the user at the time of use, such as latheringand delivery of scent for excellent in-use characteristics duringcleansing process. The term “Conditioning Stage” refers to stages inwhich the components in a personal care composition are associated withdelivery of one or more benefits during use, for example, deposition ofhydrophobic benefit phase on the skin, that provide long term benefitsafter use.

“Sagging” as used herein means the laxity, slackness, or the likecondition of skin that occurs as a result of loss of, damage to,alterations to, and/or abnormalities in dermal elastin, muscle and/orsubcutaneous fat.

“Signs of aging” include, but are not limited to, all outward visiblyand tactilely perceptible manifestations as well as any other macro ormicro effects due to skin aging. Such signs may be induced or caused byintrinsic factors or extrinsic factors (such as chronological agingand/or environmental damage). These signs may result from processeswhich include, but are not limited to, the development of texturaldiscontinuities such as wrinkles and coarse deep wrinkles, fine lines,skin lines, crevices, bumps, large pores (e.g., associated with adnexalstructures such as sweat gland ducts, sebaceous glands, or hairfollicles), or unevenness or roughness, loss of skin elasticity (lossand/or inactivation of functional skin elastin), sagging (includingpuffiness in the eye area and jowls), loss of skin firmness, loss ofskin tightness, loss of skin recoil from deformation, discoloration(including undereye circles), blotching, sallowness, hyperpigmented skinregions such as age spots and freckles, keratoses, abnormaldifferentiation, hyperkeratinization, elastosis, collagen breakdown, andother histological changes in the stratum corneum, dermis, epidermis,the skin vascular system (e.g., telangiectasia or spider vessels), andunderlying tissues (e.g., fat and/or muscle), especially those proximateto the skin.

“Skin” is divided into two main structural layers, the outer epidermisand the inner dermis, and as used herein, refers to the portion of theskin that comprises keratin-containing layers of epithelial tissuedisposed as the epidermis of mammals (e.g., humans, dogs, cats, etc.)which includes, but is not limited to, skin, mucosa, lips, hair,toenails, fingernails, cuticles, hooves, etc.

“Stage” as used herein refers to a distinguishable part in a cycle oftreatment or application of a personal care product according to thedisclosure herein. For purposes hereof, a stage need not be limited to aparticular period of time. Stages are distinct from one another in thatthe properties, most particularly the ratio of lathering phase tohydrophobic benefit phase, of a personal care composition vary betweensequential stages. Thus, in a cycle comprising three stages of treatmentor application, each stage may involve use of personal care compositionsthat vary relative to one another, for example wherein the ratio oflathering phase to hydrophobic benefit phase varies between each of thestages. In another example, in a cycle comprising three stages oftreatment or application, two of the stages may involve use of personalcare compositions that do not vary relative to one another while a thirdstage varies from the other two. In yet another example in a cyclecomprising two stages of treatment or application, each stage mayinvolve use of personal care compositions that vary relative to oneanother, for example wherein the ratio of lathering phase to hydrophobicbenefit phase varies between each of the stages.

“Stratum corneum” as used herein, refers to the outermost layer of theepithelia, or the epidermis, and is the skin structure that provides achemical and physical barrier between the body of an animal and theenvironment. The stratum corneum is a densely packed structurecomprising an intracellular fibrous matrix that is hydrophilic and ableto trap and retain water. The intercellular space is filled with lipidsformed and secreted by keratinocytes and which provide a diffusionpathway to channel substances with low solubility in water. A commonlyespoused skin metaphor portrays the stratum corneum as a brick wallwherein each brick is a corneocyte and the intercellular matrix is themortar.

“Subject” as used herein refers to an animal, such as a human, for whomproduct or composition is tested or on whom a product or composition isused in accordance with the methods described herein.

“Surfactant” as used herein means the total of all anionic, nonionic,amphoteric, zwitterionic and cationic surfactants in a phase, and isalso referred to as a “lathering phase” herein. When calculations arebased on the surfactant, water and electrolyte are excluded from thecalculations involving the surfactant, since surfactants as manufacturedtypically are diluted and neutralized.

“Test compositions” and “test agents” as used herein include andencompass purified or substantially pure compounds, as well asformulations comprising one or multiple compounds, and commerciallyavailable products. Thus, non-limiting examples of test compositionsinclude water, a pharmaceutical or cosmeceutical, a product, a mixtureof compounds or products, and other examples and combinations anddilutions thereof.

“Test surfaces” as used herein means a region of epithelia tissue whichhas been contacted with and/or by a product, such as a consumer productand/or a test product or composition, whereby the contact of the producton the epithelia tissue has resulted in some change, such as but notlimited to, physiological, biochemical, visible, and/or tactile changes,in and/or on the epithelia tissue that may be positive or negative. Insome examples, positive effects caused by product may include but arenot limited to, reduction in one or more of erythema, trans-epidermalwater loss (TEWL), discoloration of the skin, rash, dermatitis,inflammation, eczema, dandruff, edema and the like. The location of theaffected surface will depend upon the product used or the location ofsome physiological, biochemical, visible, and/or tactile change inand/or on the epithelia tissue. For example, in the case of a body washthe test surface would typically include skin on the torso orappendages, and in the case of shampoo the test surface would typicallyinclude the head/scalp, and in the case of shaving aids, the testsurface would typically include the face, underarms, or legs.

“Topical application”, “topically”, and “topical”, as used herein, meanto apply (e.g., spread, spray) the compositions used in accordance withthe present disclosure onto the surface of the skin.

“Treating” or “treatment” or “treat” as used herein includes regulatingand/or immediately improving skin cosmetic appearance and/or feel.

II. Formulating Skin Care Compositions That Improve Skin Health

As more fully described herein below, provided are methods forformulating skin care compositions based on the use of biomarker panelsfor assessing the effects of test agents on skin health. In the courseof formulating personal care compositions for the skin, selection of theactives and other composition components may be guided by theapplication of the biomarkers that are shown herein to be reflective ofskin health. Thus, in various embodiments, measurement of skin responseto test agents provides information about the biological effects of thetest agents on skin. As further described herein, positive biomarkerresults correlate well with objective physical measurements of skinhealth. Thus the biomarker panel analysis described herein can bepredictive of the ultimate benefit of an active in a skin carecomposition.

Evaluating Changes In Epithelial Tissue: Biomarkers

In some embodiments, methods are provided for evaluating changes on oneor more surfaces of epithelial tissue of a subject caused by a testproduct. The methods include measuring one or more of biomarkers thatare indicative of tissue health. In some embodiments, the methods alsoinclude measuring one or more of physical properties that are indicativeof tissue health. The methods allow for a relatively simple, efficientand quick determination of the usefulness of a test product forproviding one or more benefits to skin.

Obtaining Epithelial Tissue Samples

In accordance with some embodiments, the methods of the present involveobtaining samples of epithelial tissue to collect and analyze biomarkeranalytes. Any method suitable for obtaining epithelial tissue may beused, provided that the method obtains one or more of cellular debris,secretions from the epithelial tissue, and cells. Non-limiting examplesof suitable obtaining techniques include, application of tape, rinsingby lavage method, biopsy, swabbing, scraping, blotting and combinationsthereof. However, whichever obtaining technique is used, it must be onewhere the biomarkers obtained are those present on the surface, and/orin the epithelial tissue and not include any of the underlyingnon-epithelial tissue, such as muscle.

One suitable method of obtaining epithelial tissue is by application oftape, such as but not limited to, any type of medical tape. Thistechnique is well known in the art and is relatively simple toimplement. The technique involves application of a tape to theepithelial tissue, typically skin, which is then removed therefrom. Thebiomarker analytes obtained from the epithelial tissue and present onthe tape are then removed from the tape in any fashion that preservesthe biomarker analytes for suitable detection and measurement assays.Provided herein below are examples describing tape strip analyses.Exemplary tapes include, but are not limited to: D-squame Tape®, andSEBUTAPE®, both of which are available from CuDerm Corporation, Dallas,Tex., USA; and Transpore® tape which is available from the 3M company,of Minnesota USA.

It will be appreciated that other methods of obtaining samples ofepithelial tissue may be used, and can include not only tissue obtainedfrom a subject, but also tissue that is cultured, such as live cells.

Biomarker Analytes

Biomarker analytes are present in the test and control samples and areidentified using one or more of the techniques known in the art,examples of which are described herein in the examples. In somenon-limiting examples, biomarker analytes include inflammatorycytokines, natural moisturizing factors (NMFs), keratin 1, keratin 10,keratin 11, lipids and total protein.

Examples of inflammatory cytokines include IL1r α and IL 1α. Examples ofNMFs include amino acids, lactic acid, urea, and pyrrolidone carboxylicacid, and more particularly include Trans-Urocanic Acid, Citrulline,Glycine, Histidine, Ornithine, Proline, 2 Pyrrolidone 5 Acid, andSerine. Examples of lipids include: ceramides, including for example,NP-C18, NP-C23, NP-C26, NP-C28, NP-C30, AP-C24, AP-C26, AH-C24, AH-C26,NdS-C24, NdS-C26, EOS-C30; fatty acids, including for example C16:0,C16:1, C18:0, C18:1, C18:2; Trans-Urocanic Acid; Citrulline; Glycine;Histidine; Ornithine; Proline; 2 Pyrrolidone 5 Acid; and Serine.

The number and type of biomarker analytes detected and measured inaccordance with the provided methods therefore include thesenon-limiting examples of biomarker analytes: alpha-actinin;alpha-catenin; actin; actin binding proteins; catenins; cytokeratinstype I; cytokeratins type II; skin chymotrypsin-like enzymes;cytokeratins type I; cytokeratins type II; Desmogelin 1 and otherdesmogleins; fibronectin and fibronectin associating proteins;hyaluronic acid; involucrin; integrins; intercellular adhesionmolecules; human serum albumin; E-cadherin and classical family memberssuch, as but not limited to, desmocollin; profillagrin and its breakdown products including, but not limited to, natural moisturizationfactor and the amino acids from it; cellular retinoid binding proteins;ceramides; Cholesterol and biological modifications of cholesterolincluding, but not limited to, hormones such as cortisol andtestosterone; proteoglycans including heparan and chondroitin-6 sulfate;keratin associated proteins; loricrin; trichohyalin; Collagen, such asbut not limited to, collagens of the basement membrane such as, but notlimited to, collagens I, III and IV, and collagen-associated proteinssuch as, but not limited to, nidogen and laminin; collagenases;cornifin; calcium binding proteins, such as but not limited to S100;desmocollins; desmogleins; desmoplakin; keratohyalin; sphingolipids;total disulfide bond content for the concentration within the sample ofcysteine cross-links; inositol containing compounds; melanization signalpathways including, but not limited to, alpha-MSH, microtubules composedof tubulin and associated proteins including, but not limited to,microtubule associated protein one (MAP1) and the like; intermediatefilaments such as, but not limited to, the keratins, lamins, andvimentin and associated proteins such as but not limited to,plakoglobin; kalinin; plectin; keratohyalin granules and the proteinscontained within them; laminin; lipids; lipoproteins; nidogen;pancornulins; cornifin; keratolinin; profilin; cross-linking cellenvelope proteins; envelope pre-cursor proteins; retinoic acid bindingproteins; SPARC; small proline rich proteins (SPRR) including, but notlimited to, SPRR1, 3, and 4; spectrin and spectrin-like proteins; talin;keratinocyte transglutaminase-1 and other soluble transglutaminases;syndecan; tenascin; tensin; trichohyalin; triglycerides; tubulin;tyrosinases and their enzymatic products; vimentin; vinculin; cellulardivision markers such as, but not limited to, cyclins and cyclindependent kinases; diffipoptosis (differentiation) markers such as butnot limited to, caspase 14; and apoptosis markers and indicators suchas, but not limited to, bax and bcl-2; alpha-melanocyte stimulatoryhormone; arachidonic acid and its metabolites such as, but not limitedto, thromboxane, prostaglandins, and leucotreines; basic fibroblasticgrowth factor; vitamins; minerals (esp. Zinc, Calcium, Magnesium, etc.);cytokines and chemokines; epithelial growth factors; retinoic acid;sebocyte products; and eccrine gland products and the like.

Detection and Measurement of Biomarker Analytes

According to various embodiments, biomarker analytes are identifiedusing any of a variety of techniques known in the art for detection,including methods known in the art and described herein below fordetection of biomarkers that include but are not limited to inflammatorycytokines, natural moisturizing factors, keratin 1, keratin 10, keratin11, lipids and total protein. Thus, in various embodiments, detectiontechniques such as antibodies, nucleotide probes, highly specificchemical tags, markers, dyes, enzyme linked and other colorimetric andfluorometric probes and assays can be used to detect and measurebiomarker analytes. Kits and reagents as well as published techniquesfor biomarker detection and measurement are well known in the art.

Biomarker Panels

As described herein, a variety of biomarkers can be analyzed to assessthe effects of one or more test agents on skin. Accordingly, in variousembodiments, biomarker panels may comprise one, two or more differentbiomarkers. Representative examples of biomarker analyses and variouspanels of biomarkers are described herein in the examples section. Asdescribed herein, a variety of agents were tested for their effects onskin health, and biomarker panel testing results were analyzed forcorrelation with skin health measurements. In some examples, testcompositions including glycerin emulsions, water emulsions andalternative lipid additives were tested and demonstrated significantbiomarker changes in total protein, lipids and cytokines. In otherexamples, test compositions with fixed components in which lipid contentwas either varied or held constant demonstrated significant biomarkerchanges that evidenced persistent benefit beyond the treatment period.

These are the first known reports of cellular responses to skin careproducts. In particular, the results herein are the first known reportshowing statistically significant increases in skin lipids and reductionof inflammatory cytokines using rinse-off body care products. Theseresults are particularly surprising since the tested skin was notclassified as diseased.

As more particularly described herein below, the following observationswere made regarding the biological responses reflected by the biomarkertesting. Referring to FIG. 4 and FIG. 5 and the tables and descriptionin Example 1 hereof, we discovered that inflammatory cytokines (Log IL1ra/IL 1α) substantially decreased after 2 weeks of treatment using testcompositions in a rinse-off context. Not only were the resultssurprising since dry skin is not a diseased state, but the magnitude ofinflammation reduction was equally surprising. The decrease of Log IL1ra/IL 1α was up to 8 fold as compared to water control. Cytokinereduction trends well with barrier function improvement (TEWL)(R²=0.57).

Our results also show that total protein appears to be an early showingbiomarker. Referring now to FIG. 6 and FIG. 7 and Examples 1 and 2hereof, in testing of agents in a rinse-off context, most of the testcompositions showed meaningful differentiation from water control assoon as after 4 treatments, while the benefit continued throughout thetest period. As reported herein below, total protein correlates with dryskin improvement with R²=0.57 and 0.68 at 24 and 48 hour time points.

Referring now to Tables shown in Example 1, also in a rinse-off context,the level of natural moisturizing factors started at a lower level thanwater control, likely because of surfactant effects. However, it wasobserved over time that the surfactant effects diminished, and the testagents ultimately surpassed and trended better than the water control interms of demonstrated increase in the NMF biomarkers.

Referring now to FIG. 8, Ceramides of skin lipids showed significantenhancement with most test agents in a rinse-off context, withimprovements measured as soon as after 4 treatments compared to watercontrol.

Referring now to FIGS. 18-21, the biomarker panels including totalprotein, NMF, keratin 1, 10 and 11 and cytokines correlated well withtraditional physical measures of TEWL, dryness, corneometer andelasticity, and provided strong predictability (R²=0.65-0.74) of benefitto skin health.

In some embodiments, the methods hereof show that the quality of asubject's skin after a treatment cycle comprising at least oneconditioning stage exhibits improvement sufficient to be detected bymeasurement of one or more biomarkers that include biomarker analytesinclude inflammatory cytokines, natural moisturizing factors, keratin 1,keratin 10, keratin 11, lipids and total protein.

In some embodiments, effectiveness of treatment is evidenced bydetection of variations (or lack thereof) in at least one of biomarkerindicators or physical properties of a subject's skin after treatmentcycle, as compared to normal healthy control skin. In some embodiments,effectiveness of treatment is evidenced by no measurable variations inat least one biomarker indicator or physical property.

Improvement Indices for Biomarkers

As set forth in Examples 1, 2 and 3 hereof, indices of improvement weredetermined for each of the biomarkers in the panel described herein.

In some examples, effectiveness of treatment with a test composition isevidenced by an increase in the amount of one or more of keratin 1,keratin 10, and keratin 11, wherein the keratin improvement index isgreater than 20. For example, a keratin improvement index of from about20 to about 1000, and all points subsumed therein, would be evidence ofeffectiveness of treatment. Accordingly, a keratin improvement index of20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300,320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 520, 540, 560, 580,600, 620, 640, 660, 680, 700, 720, 740, 760, 780, 800, 820, 840, 860,880, 900, 920, 940, 960, 980, or 1000 would be evidence of effectivenessof treatment. An effective increase in the amount of one or more ofkeratin 1, keratin 10, and keratin 11 may be achieved after a suitableperiod of time after application of the product, for example, after onehour from application, or in another example, after several days ofapplication.

In some embodiments, effectiveness of treatment with a test compositionis evidenced by a decrease in the amount of one or more inflammatorycytokines, wherein the cytokine improvement index is greater than 5. Forexample, a cytokine improvement index of from about 5 to about 2000, andall points subsumed therein, would be evidence of effectiveness oftreatment. Accordingly, a cytokine improvement index of 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 40, 60, 80, 100, 120, 140,160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420,440, 460, 480, 500, 520, 540, 560, 580, 600, 620, 640, 660, 680, 700,720, 740, 760, 780, 800, 820, 840, 860, 880, 900, 920, 940, 960, 980,1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900 or 2000 wouldbe evidence of effectiveness of treatment. An effective decrease in theamount of one or more inflammatory cytokines may be achieved after asuitable period of time after application of the product, for example,after twenty four hours from application, or in another example, afterseveral days of application.

In some embodiments, effectiveness of treatment with a test compositionis evidenced by an increase in the amount of NMFs, wherein the NMFimprovement index is from about (−1) to about 25, and all pointssubsumed therein, would be evidence of effectiveness of treatment.Accordingly, a NMF improvement index of (−1), (−0.9), (−0.8), (−0.7),(−0.6), (−0.5), (−0.4), (−0.3), (−0.2), (−0.1), (−0.09), (−0.08),(−0.07), (−0.06), (−0.05), (−0.04), (−0.03), (−0.02), (−0.01), 0.00,0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 10. 1.1, 1.2, 1.3, 1.4,1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8,2.9, 3.0 to 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, or 25 would be evidence of effectiveness oftreatment. An effective increase in the amount of NMF may be achievedafter a suitable period of time after application of the product, forexample, after twenty four hours from application, or in anotherexample, after several days of application.

In some embodiments, effectiveness of treatment with a test compositionis evidenced by an increase in the amount of lipids, including ceramidesand fatty acids, wherein the ceramides improvement index is greater than5, and the fatty acids improvement index is greater than 5. For example,a ceramides improvement index of from about 5 to about 500, and allpoints subsumed therein, would be evidence of effectiveness oftreatment. Accordingly, a ceramides improvement index of 5, 6, 7, 8, 9,10, 20, 20, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160,170, 180, 190, 200, 300, 400 or 500 would be evidence of effectivenessof treatment. Likewise, a fatty acids improvement index of 5, 6, 7, 8,9, 10, 20, 20, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150,160, 170, 180, 190, 200, 300, or 400 would be evidence of effectivenessof treatment. An effective increase in the amount of one or more lipidsmay be achieved after a suitable period of time after application of theproduct, for example, after twenty four hours from application, or inanother example, after several days of application.

Measurement of Physical Properties Indicative of Skin Health

As set forth in Examples 1, 2 and 3 hereof, indices of improvement weredetermined for each of the skin health physical measurements describedherein.

In some embodiments, physical properties are measured using objectivetools and techniques that include: reduction of visual dryness,reduction in trans-epidermal water loss, increased skin hydration,increased elastic extension, increased elastic recovery, and increasedfirmness, as compared to normal healthy control skin. Also, the qualityof a subject's skin after a treatment cycle comprising at least oneconditioning stage exhibits improvement sufficient to be detected bymeasurement of one or more.

In some examples, wherein the effects of biomarkers may be correlatedwith one or more physical measures, effectiveness of treatment isevidenced by a reduction of visual dryness, wherein the reduction isgreater than 0.5 dryness units, as compared to water control. Forexample, a reduction by from about 0.5 to about 5.0 dryness units, andall points subsumed therein, would be evidence of effectiveness oftreatment. Accordingly, a reduction of 0.5, 1.0, 1.5, 2.0, 2.5, 3.0,3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10.0would be evidence of effectiveness of treatment. An effective reductionof visual dryness may be achieved after a suitable period of time afterapplication of the product, for example, after three hours fromapplication.

In some examples, effectiveness of treatment is evidenced by a reductionof trans-epidermal water loss, wherein the reduction is greater than 0.2TEWL units, as compared to water control. For example, a reduction byfrom about 0.2 to about 2.0 TEWL units, and all points subsumed therein,would be evidence of effectiveness of treatment. Accordingly, areduction of 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, or 2.0 wouldbe evidence of effectiveness of treatment. An effective reduction oftrans-epidermal water loss may be achieved after a suitable period oftime after application of the product, for example, after three hoursfrom application.

In some examples, effectiveness of treatment is evidenced by an increasein skin hydration, wherein the increase is greater than one CorneometerUnit, as compared to water control. For example, an increase of fromabout one to about 20 Corneometer Units, and all points subsumedtherein, would be evidence of effectiveness of treatment. Accordingly,an increase of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, or 20 would be evidence of effectiveness of treatment. Aneffective increase in skin hydration may be achieved after a suitableperiod of time after application of the product, for example, afterthree hours from application.

In some examples, effectiveness of treatment is evidenced by an increasein elastic extension, wherein the elastic extension improvement index isgreater than 5. For example, an elastic extension improvement index offrom about 5 to about 50, and all points subsumed therein, would beevidence of effectiveness of treatment. Accordingly, an elasticextension improvement index of 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50would be evidence of effectiveness of treatment. An effective increasein elastic extension may be achieved after a suitable period of timeafter application of the product, for example, after one hour fromapplication, or in another example, after several days of application.

In some examples, effectiveness of treatment is evidenced by an increasein elastic recovery, wherein the elastic recovery improvement index isgreater than 5. For example, an elastic recovery improvement index offrom about 5 to about 50, and all points subsumed therein, would beevidence of effectiveness of treatment. Accordingly, an elastic recoveryimprovement index of 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 would beevidence of effectiveness of treatment. An effective increase in elasticrecovery may be achieved after a suitable period of time afterapplication of the product, for example, after one hour fromapplication, or in another example, after several days of application.

In some examples, effectiveness of treatment is evidenced by an increasein skin firmness, wherein the skin firmness improvement index is greaterthan 4. For example, a skin firmness improvement index of from about 4to about 20, and all points subsumed therein, would be evidence ofeffectiveness of treatment. Accordingly, a skin firmness improvementindex of 4, 8, 12, 16, or 20 would be evidence of effectiveness oftreatment. An effective increase in skin firmness may be achieved aftera suitable period of time after application of the product, for example,after one hour from application, or in another example, after severaldays of application.

In some examples, effectiveness of treatment is evidenced by a reductionin total protein, wherein the total protein improvement index is greaterthan 5. For example, a total protein improvement index of from about 5to about 50, and all points subsumed therein, would be evidence ofeffectiveness of treatment. Accordingly, a total protein improvementindex of 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 would be evidence ofeffectiveness of treatment. An effective reduction in total protein maybe achieved after a suitable period of time after application of theproduct, for example, after twenty four hours from application, or inanother example, after several days of application.

Thus, in accordance with the various embodiments described herein,potential product formulations and actives may be tested for theireffects on skin health, wherein effectiveness of the treatment isevidenced by improvement or maintenance of at least one biomarker. Itwill be appreciated that the physical and biomarker indicators of skinquality are not limited to those identified herein and that otherbiomarkers later discovered or currently known in the art may also beassessed to determine the usefulness of a test agent for maintenance orimprovement of skin quality according to the methods hereof.

Formulating for Populations

It will be appreciated that the methods hereof are useful for benefitingusers from a variety of populations. Accordingly, also provided aremethods for developing personal care compositions and regimens oftreatment for members of various populations. The methods involvetesting proposed compositions or formulations on a plurality ofindividual subjects in a target population whereby the formulatedpersonal care composition is optimized based on the measured biomarkerresponses in the target population.

In some embodiments, the steps further include manufacturing thecomposition for the target population; and providing the composition inthe delivery article. It will be appreciated that the method may berepeated for a different target population.

III. Methods and Regimens for Treating Skin

Also provided are methods for improving the quality of skin that areevidenced by measurable improvement in one or more biomarker indicators.According to such methods, in some embodiments, the steps includedispensing from a personal care article a personal care composition thatcomprises a hydrophobic benefit phase and a lathering phase in arinse-off formulation, applying the personal care composition to asubject's skin together with water, rinsing the personal carecomposition from the subject's skin, wherein a portion of thehydrophobic benefit phase is deposited and remains on the subject's skinafter rinsing. According to such embodiments, the steps further includerepeating the steps of applying and rinsing on at least a once dailybasis over a time interval of successive days, the time interval of usesufficient to permit detection of measurable improvement in at least onebiomarker. Thus, in some specific examples, the biomarker may beselected from inflammatory cytokines, natural moisturizing factors,keratin 1, keratin 10, keratin 11, lipids and total protein. Optionally,additional characterization may be achieved by measuring one or morephysical properties to show improvement in skin condition.

According to the various embodiments, evidence of improvement based onphysical properties and biomarkers is determined using general analyticmethods known in the art. It will be appreciated, though, that thisdisclosure is the first known reported instance in which measurableimprovement in one or more skin biomarkers has been reported in thecontext of a rinse-off personal care product. Moreover, it will beappreciated that this is the first known reported instance wherein aregimen involving the use of a rinse-off composition having a variedbenefit agent profile as described herein has been employed. Thus, webelieve we are the first to report the employment of biomarker andphysical property measurements of skin to show measurable improvement inone or more properties after treatment according to the instantdisclosure. As more fully described in the examples herein, methods formeasuring physical properties and skin biomarkers have been employed todemonstrate the effectiveness of the methods hereof.

The personal care compositions used in accordance with the presentdisclosure are used in a conventional manner for use on skin, forexample, for cleansing and conditioning skin. Typically, the personalcare compositions used in accordance with the present disclosure areapplied topically to the desired area of the skin in an amountsufficient to provide effective delivery of the actives. Thecompositions can be applied directly to the skin or indirectly via theuse of an applicator pad or brush, cleansing puff, washcloth, sponge orother implement. The compositions are in some instances typicallydiluted with water prior to, during, or after topical application, andthen subsequently the skin is rinsed or wiped off, typically rinsed offof the applied surface using water or a water-insoluble substrate incombination with water.

The present disclosure is therefore in some embodiments directed tomethods of cleansing the skin through the above-described application ofthe compositions as disclosed herein. An effective amount of thecomposition for cleansing and conditioning the skin is applied to theskin, that in some examples has been wetted with water, and then rinsedoff. Such effective amounts generally range from about 1 gm to about 50gm, and from about 1 gm to about 20 gm.

In general, a typical method for cleansing and conditioning the skincomprises the steps of: a) wetting the skin with water, b) applying aneffective amount of the personal care composition to the skin, and c)rinsing the applied areas of skin with water. These steps can berepeated as many times as desired to achieve the desired cleansing andconditioning benefit.

Treatment Methods

The present disclosure is directed in some aspects to methods andregimens for improving or maintaining the quality of skin through use ofpersonal care compositions. In some aspects, the methods are useful forsustaining consumer use of a treatment for skin.

In accordance with various embodiments, the methods comprise delivery oftwo or more skin active or benefit agents to the skin of a user,particularly lathering and hydrophobic benefit phases, to provideresulting benefits from such delivery, as described herein. The personalcare compositions are formulated in various embodiments with sufficientamounts of each of the benefit agents to provide one or both ofsuperior/premium lather performance and stability and superior/premiumhydrophobic benefit phase deposition for extended conditioning. Superiorlather performance can be demonstrated via the lather volume test methoddescribed herein. Superior hydrophobic benefit phase deposition andassociated extended skin conditioning can be demonstrated via thevarious physical tests and biomarker tests described herein below.

FIG. 2 shows an exemplary profile of lathering phase to hydrophobicbenefit phase. As can be seen, one curve describes the dispensing anddelivery profile of hydrophobic benefit phase and one curve describesthe dispensing and delivery profile of lathering phase over a series ofaliquots from a representative article containing 250 ml of productaccording to the instant disclosure. Of course it will be appreciatedthat other volumes of product are contemplated, and that the ratio oflathering phase to hydrophobic benefit phase may vary depending on thepackage features and the fill profile of hydrophobic benefit phase tolathering phase. It will also be appreciated that, as described furtherherein below that additional benefit agents may be included and as such,any such additional agent may follow the profile of either thehydrophobic benefit phase or the lathering phase or may have a differentprofile. FIG. 3 shows a dispensing profile for the product exemplifiedin FIG. 2 indicating the amount of hydrophobic benefit phase by percentdelivered in the sequential aliquots as a function of dose.

In some embodiments, the compositions can comprise additional benefitagents, such as fragrances, exfoliates/desquamates, lightening and otheroptional agents as further described herein. It is contemplatedaccording to the various embodiments that the two or more skin benefitagents are delivered in varying relative quantities, as more fullydescribed herein below. It will be appreciated that additional benefitagents may be delivered together with one or the other benefit agentssuch that quantities of such additional benefit agents variessynchronously with one of the other benefit agents. It will further beappreciated that each of two, three or more benefit agents may each bedelivered in varying relative quantities that are not in synchrony withany of the other benefit agents.

The personal care compositions used in accordance with the embodimentsdisclosed herein are typically liquid or semi-liquid compositionsintended for topical application to the skin, such as the hair or skin.Thus, in some embodiments, the compositions are “rinse-off”formulations, by which is meant the product is applied topically to thehair or skin and then subsequently and immediately (i.e., withinminutes) rinsed away with water, or otherwise wiped off using asubstrate or other suitable removal means. In accordance with rinse-offcomposition embodiments, the compositions contain at least one latheringphase and at least one hydrophobic benefit phase, both of which aredescribed in greater detail hereinafter. The personal care compositionsare applied topically to the desired area of the skin or hair in anamount sufficient to provide effective delivery of the skin benefitagents to the applied surface, or to otherwise provide effective skinconditioning benefits. The compositions can be applied directly to theskin or indirectly via the use of a cleansing puff, washcloth, sponge orother implement. The compositions are in some embodiments diluted withwater prior to, during, or after topical application.

Examples of application and use of some embodiments of personal carecompositions are provided herein. Likewise, examples describing themethods for characterizing the premium benefits of the compositions, andfor preparing and packaging embodiments of the compositions are providedin the examples sections hereof.

In various embodiments, the methods and regimens comprise use of apersonal care composition that comprises at least two skin active orbenefit agents, typically a lathering phase (surfactant) and ahydrophobic benefit phase (lipid), wherein the amounts (ratio, byweight) of lathering phase to hydrophobic benefit phase are constant orvary over the course of the regimen. The composition is used over aperiod of time, alternately referred to as a treatment cycle ortreatment time that includes one, two or more sequential stages. Itshould be understood that the term “stage,” as used to describe themethods herein, is intended to be non-limiting with respect to time orsequence of the steps of a treatment cycle. In some embodiments, thetreatment cycle includes three stages.

In some embodiments, the treatment cycle comprises at least one stagewhere the ratio of the lathering phase to the hydrophobic benefitessentially does not vary. It will be appreciated that a treatment cyclecan include two, three, four or more stages, and that the ratio oflathering phase to hydrophobic benefit phase may be the same throughoutall of the stages, or the ratio may vary between the stages though theratio remains constant throughout a singe stage.

In alternate embodiments, the ratio of the lathering phase to thehydrophobic benefit phase varies from one stage to the next stage,wherein in some embodiments the ratio of lathering phase to hydrophobicbenefit phase is greater in a first stage than in a second stage. Inother embodiments, the ratio of lathering phase to hydrophobic benefitphase is lower in a first stage than it is in a second stage. In someembodiments comprising three stages, the ratio of lathering phase tohydrophobic benefit phase is higher in a first stage, declines in asecond stage, and increases in a third stage. It will be appreciatedthat a treatment cycle can include two, three, four or more stages, andthat the ratio of lathering phase to hydrophobic benefit phase can varyin a variety of ways between each of the sequential stages, as isdescribed more fully herein below. In accordance with the variousembodiments, the methods include the steps of applying the compositionto a subject's skin on a daily basis for a period of days.

According to a representative embodiment comprising a treatment cycle ofthree stages, a first stage is a premium experience stage in which ahigh lathering phase (surfactant) is used that provides a premium userexperience through high lather. Thus, in this first stage, the ratio oflathering phase to hydrophobic benefit phase is high relative to thefollowing stage. According to this representative embodiment, as useprogresses into a second stage, a high lipid “plateau” providesconditioning through high hydrophobic benefit phase content. Thus, inthis second stage, the ratio of lathering phase to hydrophobic benefitphase is low relative to a first stage. And in a third stage a highlathering phase (surfactant) is used that provides a premium userexperience through high lather. Thus, in this third stage, the ratio oflathering phase to hydrophobic benefit phase is high relative to theprevious stage. It will be appreciated that the number and sequentialorder of premium experience and conditioning stages may vary. Asmentioned above, in some embodiments a treatment period may compriseonly two stages, or it may comprise three or more stages. Irrespectiveof the number and order of stages, a treatment cycle is characterized insome embodiments as comprising in any order at least one premiumexperience stage and one conditioning stage.

According to the various embodiments, personal care compositions providea lathering phase that produces a lather volume. In some embodiments thelather volume of the composition is greater than from about 800 ml to1500 ml, as tested according to the Lather Volume method describedherein. It will thus be appreciated by those in the art that inaccordance with the described method, the lather volume provided by apersonal care composition may be greater than from about 800, 850, 900,950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, and1500 or more ml as well as all points subsumed therein. Of course itwill be appreciated that in some embodiments, particularly with respectto embodiments and stages wherein the ratio of lathering phase tohydrophobic benefit phase is low, that the lather volume will be loweror substantially lower than the above stated ranges. In some embodimentsthe lather volume may be greater than the above ranges, such as fromabout 1500 to 1750, about 1750 to 1900, about 1900 to 2000 or more ml,as well as all points subsumed therein. It will also be appreciated thatother methods described or otherwise known in the art may be used tocharacterize lather and lather volume and that the description herein isnot limiting, such that the lather properties of the compositions usedas described herein may be described in other terms.

In accordance with the provided methods, the composition is provided ina delivery article that is adapted for use in accordance with apredetermined time of treatment or a predetermined approximate number ofinstances of treatment, or both. Thus, in some embodiments, the deliveryarticle is adapted to deliver sufficient composition for one or two ormore treatment cycles. In some embodiments, the delivery article isadapted to deliver the composition for each treatment cycle in anapproximate number of aliquots or units. In such embodiments, thealiquots may be the same in volume or may vary. In some embodiments thenumber of aliquots or units to be dispensed per stage or in a deliveryarticle is a predetermined number that defines the approximate number ofinstances of use, either in days, weeks or months.

In accordance with some embodiments of the methods, the composition isapplied on a daily basis. A delivery article as described above mayoptionally be used for delivery of the composition. It will beappreciated that treatment times and frequency may vary based upon theuser, and as such, treatment may be on a less than daily basis, or maybe more often. In other embodiments, the treatments may be lessfrequent, for example weekly or monthly, or in some other interval oftime.

As described herein above with respect to the stratum corneum, that is,the outermost layer of the skin, that the cycle of cell turnover in theepidermis from the basal stratum to the stratum corneum is on the orderof about a month, or from about 27 to 30 days. In addition, the averagesloughing (desquamation) time of cells from the stratum corneum is onthe order of about two weeks, or from about 12-14 days. It has beenappreciated with respect to the methods hereof, that treatment cyclesadapted to the biology of the skin are surprisingly effective inimproving and maintaining the normal function and healthy quality ofskin, as more fully described herein with respect to thecharacterization of skin properties. Thus, treatment cycles which spanthe typical epidermal cell cycle (for keratinocytes) and which includeat least one conditioning stage of about a week have been demonstratedto be effective, as described herein.

In some embodiments of the methods hereof the composition is appliedthrough a treatment cycle in a time interval of about thirty days. Forexample, a first stage of the treatment cycle may be from about 3 to 7days, a second stage of the treatment cycle may be from about 6 to 14days, and a third stage of the treatment cycle may be from about 6 to 14days. In another example, a first stage of the treatment cycle may befrom about 2 to 5 days, a second stage of the treatment cycle may befrom about 3 to 7 days, and a third stage of the treatment cycle may befrom about 14 to 21 days. In accordance with these examples, in someembodiments the first and third stages are premium experience stages,wherein the delivery of lathering phase and associated lather volume ishigh as compared with the second, conditioning stage, wherein thedelivery of hydrophobic benefit phase is high.

It will be appreciated that according to the methods, a treatment cyclemay be repeated two or more times. According to various embodiments,repeated treatment cycles may have the same number and order of stagesand may be of the same length of time. Of course, in other embodiments,sequential treatment cycles may each have different numbers and ordersof stages and may be of varying lengths of time. In accordance with arepresentative embodiment of a treatment cycle as described above,wherein the cycle is about 30 days or one month, the treatment cycle maybe repeated one or more times. Thus, in a series of two treatmentcycles, there are six stages ordered as premium experience,conditioning, premium experience, premium experience, conditioning,premium experience. As in the example described above, the number ofdays of each cycle may be as described, or the number of days may be asdescribed in alternate embodiments described below.

In other embodiments, the composition is applied through a treatmentcycle in a time interval of about fifty days. In one example, a firststage of the treatment cycle may be from about 3 to 7 days, a secondstage of the treatment cycle may be from about 10 to 28 days, and athird stage of the treatment cycle may be from about 14 to 20 days. Inother embodiments, the composition is applied through a treatment cyclein a time interval of about fifty-six days. In one example, a firststage of the treatment cycle may be from about 2 to 7 days, a secondstage of the treatment cycle may be from about 3 to 28 days, and a thirdstage of the treatment cycle may be from about 6 to 21 days.

Optionally, the composition used in accordance with the methods maycomprise an additional benefit agent, non-limiting examples of whichinclude vitamins, vitamin derivatives, sunscreens, desquamation actives,anti-wrinkle actives, anti-atrophy actives, anti-oxidants, skin soothingagents, skin healing agents, skin lightening agents, skin tanningagents, anti-acne medicaments, essential oils, sensates, pigments,colorants, pearlescent agents, interference pigments, particles,hydrophobically modified non-platelet particles and combinationsthereof. In one example the additional benefit agent is a fragrance. Inaccordance with this example, some embodiments the fragrance agent isdelivered with the lathering phase such that the premium experiencestage is further characterized by delivery of fragrance. Of course, inother embodiments, the additional agent, such as a fragrance agent, maybe delivered with the hydrophobic benefit phase so as to provideenhanced experience during the conditioning stage. And of course in yetother embodiments, the additional benefit agent, such as a fragranceagent, may be delivered in with both the lathering and the hydrophobicbenefit phases. In some embodiments, more than one additional benefitagents, such as two fragrance agents, may be included and the amount orpresence of each may vary through the stages. According to the variousembodiments comprising additional benefit agent(s), the additionalbenefit agent(s) may be provided in a fixed amount or concentration, orin amounts that vary across the stages, or that vary with one or theother of the lathering and hydrophobic benefit phases, or that varyseparately from each of the other benefit agents. Other benefit agentsand materials as described herein and those known in the art may also beused with respect to representative composition embodiments describedherein. Likewise, other formulation components, including other andadditional lathering/surfactant agents and hydrophobic benefit phasesmay be selected as described herein. Additional benefit agents may beprovided with either or both the lathering phase and the hydrophobicbenefit phase. Examples of some specific benefit agents includeexfoliating agents, niacinamide, vitamin E (tocopherol or tocotrieneol),collagen.

The following examples further describe and demonstrate embodimentswithin the scope of the present disclosure. The examples are givensolely for the purpose of illustration and are not to be construed aslimitations of the present disclosure, as many variations thereof arepossible without departing from the spirit and scope hereof.

IV. Examples

Example 1: Clinical Study: Evaluation of Skin Indicator Response andCytokine, Natural Moisturizing Factor, Total Protein and LipidBiomarkers; Correlations Between Biomarkers and Measures of Skin Health

The clinical study design was a leg controlled application test (LCAT)protocol for body wash, representative methods are described hereinbelow.

Clinical design and biomarker analysis: _Human subjects were screenedfor dry skin score at 2.5 or higher, in accordance with the drynessgrading procedure described herein below. A cohort of 30 subjects wasselected for each treatment. All subjects were pre-conditioned withOlay® soap bar for 7 days followed by 1 application/day for 3 weeks and2 day regression. Measurements included dry skin grade, corneometer,TEWL, cutometer, and tape strips to obtain biomarker analytes. Thetreatment design is shown in TABLE 1. The formulations for each of thecompositions A, B, C, D, E, F, and G are provided in TABLES 2, 3, 4, 5,6, and 7 below.

TABLE 1 CLINICAL DESIGN Leg Lipid Phase A Water B Glycerin emulsion(1.5% GMO, 68.5% G2218Pet, 30% glycerin) + surfactant (50:50) C Wateremulsion (1.5% GMO, 68.5% G2218Pet, 30% water) + surfactant (50:50) DWater emulsion (1.5% GMO, 68.5% G2218Pet, 30% water) + glycerin (30%)surfactant (50:50) G Sefose lipid (8.0% sefose 1618H, 2.0% sefose1618U,90.0% G2218Pet) + surfactant (50:50)

TABLE 2 SURFACTANT CTFA NAME Percent Composition Water 31.796%  SodiumLauryl Sulfate 28.550%  Sodium Lauroamphoacetate 15.940%  SodiumTrideceth Sulfate 12.740%  Sodium Chloride 4.750% Fragrance (PashminaPCC) 1.820% Trideceth-3  2.00% Methylchloroisothiazolinone, 0.091%Methylisothiazolinone Citric Acid 0.895% Guar Hydroxypropyltrimonium 0.60% Chloride Xanthan Gum 0.220% Sodium Benzoate 0.200% PEG-90M 0.150%Disodium EDTA 0.150% Sodium Hydroxide 0.098%

TABLE 3 GLYCERIN (30%) SURFACTANT CTFA NAME Percent Composition Water1.796% Glycerine 30.000%  Sodium Lauryl Sulfate 28.550%  SodiumLauroamphoacetate 15.940%  Sodium Trideceth Sulfate 12.740%  SodiumChloride 4.750% Fragrance 1.820% Trideceth-3  2.00%Methylchloroisothiazolinone, 0.091% Methylisothiazolinone Citric Acid0.895% Guar  0.60% Hydroxypropyltrimonium Chloride Xanthan Gum 0.220%Sodium Benzoate 0.200% PEG-90M 0.150% Disodium EDTA 0.150% SodiumHydroxide 0.098%

TABLE 4 1.5% GMO, 68.5% G-2218 PETROLATUM, 30% GLYCERIN LEG B (+SURFACTANT IN TABLE 2) Percent CTFA NAME Composition Glycerol Monooleate(GMO) 1.5000 Petrolatum-Low Kaydol 68.5000 Kosher Superol Glycerine30.0000 100.0000

TABLE 5 1.5% GMO, 68.5% G-2218 PETROLATUM, 30% WATER LEG C (+ SURFACTANTIN TABLE 2) Percent CTFA NAME Composition Glycerol Monooleate 1.5000Petrolatum-Low Kaydol 68.5000 Water 30.0000

TABLE 6 1.5% GMO, 68.5% G-2218 PETROLATUM, 30% WATER LEG D (+ SURFACTANTIN TABLE 3) Percent CTFA NAME Composition Glycerol Monooleate 1.5000Petrolatum-Low Kaydol 68.5000 Water 30.0000

TABLE 7 90.0% G-2218 PETROLATUM, 8.0% SEFOSE 1618H, 2.0% SEFOSE LEG G (+SURFACTANT IN TABLE 2) Percent CTFA NAME Composition .alpha.-D- 8.0000Glucopyranoside, .beta.- D-fructofuranosyl, octadecanoate (Hardened SefaSoyate) Fatty acids C-16-18 and 2.0000 C18-unsaturated esters withSucrose Petrolatum-Low Kaydol 90.0000 100.0000

Biomarker analysis: Quantitative biomarker analysis included totalproteins, cytokines, NMFs, and skin lipids. The tape strips collectedfrom the clinical study were extracted with buffer solutions. Biomarkersin the buffer solutions were quantified via multiplex ELISA and LC/MS/MSand the results were subjected to statistical analysis.

Biomarker Results and Discussion

Cytokines: Cytokines are immunomodulating agents. Specific cytokinesevaluated included interleukin 1, alpha (IL 1α) and interleukin 1receptor antagonist (IL 1ra). It is found in the academic literaturethat the ratio of IL 1ra and IL 1α is a good indicator for skininflammation and irritation.

It was quite surprising and unexpected to discover that certain of thecompositions tested according to the instant methods significantlyreduce log (IL 1ra/IL 1α) vs. the water control after only 14 days, asshown in FIG. 4, particularly in view of the fact that according toconventional wisdom at the time of the instant application, dry skin isnot at diseased state. As shown, the magnitude of the log (IL 1ra/IL 1α)is increased up to eightfold.

We analyzed data from physical measurement studies and biomarkeranalysis for correlations between them, and found that inflammatorycytokine reduction correlates well with the improvements in traditionalclinical measurement of dryness, TEWL and corneometer, as shown in FIG.4 and FIG. 5. This disclosure is the first known report of measurableinflammatory benefits from a body wash composition based on measurementof inflammatory cytokines.

Total proteins: This marker measures protein levels, which in accordancewith the method of this study, were captured on the tape. Morecohesiveness in healthy stratum corneum produces less protein on thetape strip, while high proteins are predicted to be obtained as a resultof less cohesiveness in damaged skin. Referring now to TABLE 8B hereinbelow, results are shown for total protein of 6 tape strips for eachtest product at 24 hours post treatment time point over a treatmentperiod of 3 weeks and 2 day regression (no treatment). Almost all testproducts show significant or directional improvement vs. water control(A) at all time points. The products start differentiating from watercontrol as soon as after 4 treatments and the effect reaches the highestpoint at 3 weeks (22 days). All test products are significantly betterthan water control after 21 treatments (22 day). Applying a correlationanalysis as represented in FIG. 6 and FIG. 7, total protein trendsconsistently with skin dryness improvement at both 24 and 48 hours. Thatis, dryness improvement increases as total protein decreases. In otherwords, skin cohesiveness enhancement is achieved with effective dry skinimprovement.

Natural moisturizing factors: Natural moisturizing factor is acollection of water-soluble compounds that are predominantly found inthe stratum corneum. These compounds compose approximately 20-30% of thedry weight of a corneocyte. NMF components absorb water from theatmosphere and combine it with their own water content allowing theoutermost layers of the stratum corneum to stay hydrated despiteexposure to the elements. Referring now to TABLE 8C, results are shownfor NMF. NMF measurements in this study included amino acids, lacticacid, urea, and pyrrolidone carboxylic acid. Because NMF components arewater soluble, they are easily leached from the cells with watercontact, which we propose is why NMF in water control (A) decreasesthrough repeated treatment. NMF levels in products before 14 days arelower than water control. We propose this effect may be a result ofsurfactants more effectively washing NMF out. Interestingly, most testproducts reach the same level and some show improvement over watercontrol, and in particular, the glycerin-GMO-Pet emulsion (B) showsdirectional advantage over water control.

Lipids: The intercellular lipids of the human stratum corneum are uniquein composition and quite different from the lipids typically found inbiological membranes. They are ideally suited to the formation ofpermeability barrier because of their high melting point and polarity,which lead to the formation of water-resistant lipid bilayers. The majorlipids of the human stratum corneum are ceramides, cholesterol and fattyacids, comprising about 50%, 25% and 10% respectively of the total lipidmass. Referring now to TABLES 8D and 8E, results are shown forceramides. The ceramides measured and detected include NP-C18, NP-C23,NP-C26, NP-C28, NP-C30, AP-C24, AP-C26, AH-C24, AH-C26, NdS-C24,NdS-C26, EOS-C30. The test products show significant increases in allindividual ceramides markers compared to water control after 2 weeks oftreatment. Referring now to TABLE 8E, significant increases of theproducts in fatty acids are observed in all markers measured (C16:0,C16:1, C18:0, C18:1, C18:2). The increase of ceramides trends well withTEWL decrease after 3 week treatment, as shown in FIG. 8.

Tables 8A-8H show detailed clinical results summary data, and include:

TABLE 8A CYTOKINE, Log(IL 1ra/IL 1α) Statistical Grouping Sample (95%Adjusted Standard Attribute Evaluation Size Treatment confidence) MeanError Log(IL1ra/IL1Alpha) Day 1, 37 [A] Water ab −0.849 0.173 Baseline36 [B] Glycerin emulsion + a −0.996 0.174 surfactant 37 [C] WaterEmulsion + ab −0.879 0.173 surfactant 38 [D] Water Emulsion + b −0.7450.172 glycerin surfactant 37 [G] Sefose lipid + a −0.991 0.173surfactant Log(IL1ra/IL1Alpha) 24 Hr Post 36 [A] Water c −0.392 0.113Wash 13 36 [B] Glycerin emulsion + ab −0.703 0.113 (14.0) surfactant 37[C] Water Emulsion + ab −0.715 0.112 surfactant 38 [D] Water Emulsion +a −0.950 0.111 glycerin surfactant 37 [G] Sefose lipid + ab −0.690 0.112surfactant Log(IL1ra/IL1Alpha) 24 Hr Post 37 [A] Water c −0.046 0.120Wash 21 36 [B] Glycerin emulsion + ab −0.803 0.122 (22.0) surfactant 37[C] Water Emulsion + b −0.674 0.120 surfactant 37 [D] Water Emulsion + a−0.939 0.121 glycerin surfactant 37 [G] Sefose lipid + ab −0.737 0.121surfactant

TABLE 8B TOTAL PROTEIN (SQUAMESCAN 850 UNIT) Statistical Grouping Sample(95% Adjusted Standard Attribute Evaluation Size Treatment confidence)Mean Error Sum of First 6 Day 1, 36 [A] Water a 117.44 3.126 Tape StripsBaseline 36 [B] Glycerin emulsion + a 115.95 3.125 surfactant 37 [C]Water Emulsion + a 115.64 3.094 surfactant 37 [D] Water Emulsion + a114.95 3.094 glycerin surfactant 37 [G] Sefose lipid + a 112.30 3.094surfactant Sum of First 6 24 Hr Post 37 [A] Water b 132.86 2.648 TapeStrips Wash 21 36 [B] Glycerin emulsion + a 124.26 2.645 (22.0)surfactant 37 [C] Water Emulsion + a 121.13 2.618 surfactant 37 [D]Water Emulsion + a 123.92 2.646 glycerin surfactant 37 [G] Sefoselipid + a 122.36 2.622 surfactant

TABLE 8C NATURAL MOISTURIZING FACTORS (NMF), Log(Sum NMF/solubleprotein) Statistical Grouping Sample (95% Adjusted Standard AttributeEvaluation Size Treatment confidence) Mean Error Log(Sum Day 1, 36 [A]Water a 3.745 0.006 NMF/soluble protein) Baseline 36 [B] Glycerinemulsion + a 3.745 0.006 surfactant Log(Sum 24 Hr Post 36 [A] Water a3.729 0.007 NMF/soluble protein) Wash 13 36 [B] Glycerin emulsion + a3.715 0.007 (14.0) surfactant Log(Sum 48 Hr Post 37 [A] Water ab 3.7350.005 NMF/soluble protein) Wash 21 35 [B] Glycerin emulsion + b 3.7460.005 (23.0) surfactant

TABLE 8D CERAMIDES, Log(SumCeramide/soluble protein) StatisticalGrouping Sample (95% Adjusted Standard Attribute Evaluation SizeTreatment confidence) Mean Error Log(SumCeramide/ Day 1, 36 [A] Water a0.841 0.048 soluble protein) Baseline 36 [B] Glycerin emulsion + a 0.8700.048 surfactant 37 [C] Water Emulsion + a 0.833 0.047 surfactant 37 [D]Water Emulsion + a 0.807 0.047 glycerin surfactant 37 [G] Sefose lipid +a 0.831 0.047 surfactant Log(SumCeramide/ 24 Hr Post 36 [A] Water ab0.846 0.046 soluble protein) Wash 13 36 [B] Glycerin emulsion + ab 0.8630.045 (14.0) surfactant 36 [C] Water Emulsion + abc 0.880 0.045surfactant 38 [D] Water Emulsion + bc 0.958 0.045 glycerin surfactant 37[G] Sefose lipid + c 0.991 0.045 surfactant Log(SumCeramide/ 24 Hr Post37 [A] Water a 0.686 0.046 soluble protein) Wash 21 36 [B] Glycerinemulsion + ab 0.780 0.046 (22.0) surfactant 37 [C] Water Emulsion + b0.880 0.046 surfactant 37 [D] Water Emulsion + b 0.811 0.046 glycerinsurfactant 37 [G] Sefose lipid + b 0.847 0.046 surfactant

TABLE 8E FATTY ACIDS, Log(SumFatty acids/soluble protein) StatisticalGrouping Sample (95% Adjusted Standard Attribute Evaluation SizeTreatment confidence) Mean Error Log(SumFatty Day 1, 36 [A] Water a2.361 0.067 acids/soluble Baseline 36 [B] Glycerin emulsion + b 2.4960.067 protein) surfactant 37 [C] Water Emulsion + ab 2.448 0.066surfactant 37 [D] Water Emulsion + ab 2.460 0.066 glycerin surfactant 37[G] Sefose lipid + ab 2.438 0.066 surfactant Log(SumFatty 24 Hr Post 37[A] Water a 2.341 0.056 acids/soluble Wash 21 36 [B] Glycerin emulsion +b 2.494 0.056 protein) (22.0) surfactant 37 [C] Water Emulsion + b 2.5520.055 surfactant 37 [D] Water Emulsion + b 2.534 0.056 glycerinsurfactant 37 [G] Sefose lipid + b 2.514 0.055 surfactant

TABLE 8F EXPERT DRYNESS GRADES Statistical Grouping Sample (95% AdjustedStandard Attribute Evaluation Size Treatment confidence) Mean ErrorExpert dryness Day 1, 37 [A] Water a 2.992 0.049 grade Baseline 36 [B]Glycerin emulsion + a 3.045 0.050 surfactant 38 [C] Water Emulsion + a3.021 0.049 surfactant 37 [D] Water Emulsion + a 3.064 0.049 glycerinsurfactant 37 [G] Sefose lipid + a 3.011 0.049 surfactant Expert dryness24 Hr Post 26 [A] Water c 3.369 0.116 grade Wash 13 30 [B] Glycerinemulsion + ab 2.997 0.110 (14.0) surfactant 29 [C] Water Emulsion + a2.793 0.112 surfactant 30 [D] Water Emulsion + a 2.823 0.110 glycerinsurfactant 27 [G] Sefose lipid + a 2.827 0.115 surfactant Expert dryness24 Hr Post 37 [A] Water b 3.329 0.095 grade Wash 21 36 [B] Glycerinemulsion + a 2.840 0.096 (22.0) surfactant 37 [C] Water Emulsion + a2.853 0.095 surfactant 37 [D] Water Emulsion + a 2.909 0.095 glycerinsurfactant 37 [G] Sefose lipid + a 2.941 0.095 surfactant

TABLE 8G CORNEOMETER RESULTS Statistical Grouping Sample (95% AdjustedStandard Attribute Evaluation Size Treatment confidence) Mean ErrorCorneometer Day 1, 37 [A] Water a 18.806 0.691 Baseline 36 [B] Glycerinemulsion + a 18.981 0.699 surfactant 37 [C] Water Emulsion + a 18.6800.691 surfactant 38 [D] Water Emulsion + a 19.136 0.684 glycerinsurfactant 37 [G] Sefose lipid + a 18.613 0.691 surfactant Corneometer24 Hr Post 26 [A] Water a 18.384 0.695 Wash 13 30 [B] Glycerinemulsion + b 20.598 0.655 (14.0) surfactant 29 [C] Water Emulsion + b21.995 0.665 surfactant 30 [D] Water Emulsion + b 21.204 0.655 glycerinsurfactant 27 [G] Sefose lipid + b 21.223 0.687 surfactant Corneometer24 Hr Post 37 [A] Water a 17.635 0.660 Wash 21 36 [B] Glycerinemulsion + bc 21.973 0.667 (22.0) surfactant 37 [C] Water Emulsion + c22.499 0.661 surfactant 37 [D] Water Emulsion + bc 21.588 0.660 glycerinsurfactant 37 [G] Sefose lipid + bc 21.492 0.661 surfactant

TABLE 8H TEWL Statistical Grouping Sample (95% Adjusted StandardAttribute Evaluation Size Treatment confidence) Mean Error TEWL readingsDay 1, 36 [A] Water a 5.547 0.210 (Avg. of 2 probes) Baseline 36 [B]Glycerin emulsion + a 5.489 0.210 surfactant 37 [C] Water Emulsion + a5.366 0.208 surfactant 38 [D] Water Emulsion + a 5.443 0.206 glycerinsurfactant 37 [G] Sefose lipid + a 5.450 0.208 surfactant TEWL readings24 Hr Post 26 [A] Water b 6.905 0.270 (Avg. of 2 probes) Wash 13 30 [B]Glycerin emulsion + a 5.314 0.255 (14.0) surfactant 29 [C] WaterEmulsion + a 5.747 0.259 surfactant 30 [D] Water Emulsion + a 5.6090.255 glycerin surfactant 27 [G] Sefose lipid + a 5.807 0.266 surfactantTEWL readings 24 Hr Post 37 [A] Water d 7.200 0.181 (Avg. of 2 probes)Wash 21 36 [B] Glycerin emulsion + a 5.749 0.181 (22.0) surfactant 37[C] Water Emulsion + b 6.176 0.179 surfactant 37 [D] Water Emulsion + b6.134 0.179 glycerin surfactant 37 [G] Sefose lipid + b 6.218 0.179surfactant

Example 2: Clinical Study: Evaluation of Skin Indicator Response andKeratin and Total Protein Biomarkers; Correlations Between Biomarkersand Measures of Skin Health

A study was undertaken to evaluate the response of a variety of skinindicators using an array of different personal care compositions andwater. The clinical study design was a leg controlled application test(LCAT) protocol for body wash used to evaluate the beneficial effects ofpersonal care products on dry leg skin. Leg wash studies are designed toapproximate consumer-relevant exposure levels, e.g. washing frequency.The technique used in this study is a modification of a publishedprocedure (Ertel, et al, 1999).

The study included a product having a variable lipid profile, whereinthe ratio of surfactant to lipid (lathering phase to hydrophobic benefitphase) varied continuously between three stages according to the instantdisclosure. The study also included a commercially available producthaving a constant lipid profile. As further described herein below, thecompositions used in the study included Inventive Example B andComparative Example C, as shown in Table 9.

TABLE 9 EXAMPLES OF CONTINUOUS AND VARIABLE LIPID COMPOSITIONS Code B -Continuous Code C - Lipid (55% Variable Lipid surfactant to (shown inFIG. 45% lipid) 9) SURFACTANT PHASE Sodium Lauroamphoacetate^(1.) 4.94.9 Sodium Trideceth Sulfate^(2.) 8.4 8.4 Sodium Lauryl Sulfate 8.4 8.4Trideceth-3^(3.) 2.0 2.0 Sodium Chloride 4.75 4.75 Guarhydroxypropyltrimonium 0.6 0.6 chloride^(4.) Polyethyleneoxide^(5.) 0.150.15 Xanthan gum^(6.) 0.2 0.2 Hollow microspheres^(7.) 0.3 0.3 Methylchloro isothiazolinone and 0.0005 0.0005 methyl isothiazolinone^(8.)EDTA^(9.) 0.15 0.15 Sodium Benzoate 0.2 0.2 Citric Acid, titrate pH =5.7 ± 0.2 pH = 5.7 ± 0.2 Perfume 1.3 1.3 Water Q.S. Q.S. BENEFIT PHASEPetrolatum^(10.) 70 70 Mineral Oil^(11.) 30 30 ^(1.)Available fromCognis Chemical Corp. ^(2.)sulfanated to >95% sulfate from ICONOL ®TDA-3 available from BASF Corp., ^(3.)ICONOL ® TDA-3 available from BASFCorp., ^(4.)N-HANCE ® 3196 Polymer from Aqualon of Wilmington, DE,^(5.)POLYOX ™ WSR-301 available from DOW ® Chemical Corp., ^(6.)KELTRO ™1000 available from CP Kelco, ^(7.)EXPANCEL ® microspheres availablefrom 091 WE40 d24, Akzo Nobel, ^(8.)KATHON ® CG available for Rohm &Haas, ^(9.)DISSOLVINE ® NA 2x available from Akzo Nobel, ^(10.)G2218petrolatum from Sonneborn, ^(11.)HYDROBRITE ® 1000 White Mineral Oilavailable from Sonneborn.

FIG. 9 shows a representative dispensing profile for the lipid(hydrophobic benefit phase) in the variable lipid product of the presentdisclosure. The profile indicates with arrows the treatment days atwhich certain measurements as described herein were made. Thus, thetreatment days are shown with reference to the initiation of treatmentafter the preconditioning interval; day 1 as shown on FIG. 9 correspondsto study day 8, and so on.

The study was 29 days in duration, with a 7 day interval ofpreconditioning, 21 days of treatment and 1 regression day. Skin wasanalyzed at various points from the beginning through the end of thestudy period. The objective of the study was to characterize the dryskin improvement profile of several body wash prototypes and to generatesamples to assess treatment's effects on stratum corneum physical andbiomarker indicators. After the 7-day preconditioning stage, subjectsreturned to the test facility to have the skin on their lower legsevaluated by an expert grader. Only subjects which exhibited sufficientdryness on all of the treatment sites qualified to continue into thetreatment stage. Technicians treated each qualified subject's lower legsin a controlled manner with the assigned treatments once daily for 21days. Subjects' legs were visually evaluated for dryness and redness atseveral pre and post-treatment times as outlined in the following studyschedule. Non-invasive instrumental measurements of stratum corneumhydration (Corneometer 825), barrier function (Dermalab TEWL (transepidermal water loss)), and viscoelasticity (Cutometer) were made on thetreatment sites following visual evaluations.

Treatment Stage Procedure: Before initial grading on Study Day 8, testfacility personnel marked off the leg application areas [two 70 cm²areas (7 cm across×10 cm down)] on the outer aspect of the subjects'lower legs using a template and laboratory marking pen (corner bracketsare sufficient to delineate each area). Trained clinical assistantstreated each subject's legs according to the procedure outlined in theTreatment Procedure. In general, the following should be noted: Theprocedure was conducted once each day for 21 consecutive days. The bodywash products were applied using puffs (personal cleaning implements).The puff treatment procedure for all puffs was conducted daily after allproduct treatments were completed on each subject (except on the finalday of treatment.).

Evaluations: At each evaluation, subjects acclimated for a minimum of 30minutes in a room with the environment maintained at 70° F.±2 and 30-45%relative humidity prior to visual grading and non-invasive instrumentalmeasurements being made on their legs.

Visual Grading: Each subject's lower legs were visually evaluated by aqualified grader for dryness and redness at baseline (Study Day 8, priorto the first treatment) as a prerequisite for qualification into thetreatment stage. Measurements were made thereafter on study days 10, 12,21, 28 and 29, at approximately 3 hours post treatment. Referring toFIG. 10, comparative results according to the study are shown.

Corneometer Skin Capacitance: Non-invasive skin capacitance measurementswere taken in duplicate on each site of the subjects' legs after everyvisual grading during the study using a Corneometer CM825 instrument.Data was recorded electronically using the Sponsor's direct data entryand data capture programs. The same instrument and operator were usedthroughout the study. Referring to FIG. 11, comparative resultsaccording to the study are shown.

Trans-epidermal water loss: TEWL was measured with the DermaLab®Evaporimeter equipped with dual probes. Each measurement consists ofreadings collected for 60 seconds with the mean of the last 20 secondsrecorded from both probes (Channel A and Channel B). One measurement wastaken at each treatment site and recorded on DCF 2 (DermaLab TEWLMeasurements Log) on each evaluation day for both probes as Channel Aand Channel B, respectively. The same instrument and operator were usedthroughout the study. These measurements were made according toprocedures outlined in accordance with published guidelines.Measurements were taken 8 times during the course of the study on studydays 10, 12, 21, 28 and 29, at approximately 3 hours post treatment.Referring to FIG. 12, comparative results according to the study areshown.

Cutometer Measurements of Elasticity: Reapplication of methods typicallyused for facial skin in a leave-on context was used with a cutometer onlegs in a rinse-off personal care composition context. Non-invasive skinviscoelasticity measurements were taken with a Cutometer SEM 575equipped with an 8 mm probe. Data was recorded electronically using thedata capture program accompanying the instrument. Two Cutometerinstruments were used due to the number of subjects enrolled in thestudy. Subjects were assigned to the same instrument throughout thestudy on the basis of there subject number. The same instruments andoperators were used throughout the study. These measurements were madeaccording to the procedures outlined in the Sponsor's instrument SOPs orpublished guidelines. Measurements were taken 5 times during the courseof the study on study days 10, 12, 21, 28 and 29, at approximately 3hours post treatment. Referring to FIG. 13, FIG. 14 and FIG. 15,comparative results according to the study are shown.

Tape Stripping: Tape stripping was performed throughout the study fordry skin sampling. D-Squames was always collected following all otherevaluations scheduled to take place at the same time point. Clinicalassistants wore disposable gloves while collecting D-Squames. At eachcollection time point a series of 6 D-Squames were used to sample thesame spot within the treatment area. The technician used forceps toplace a D-Squames sampling disc toward the edges of each site (away fromthe region being evaluated by other instrumentation) and appliedpressure using the D-Squames disc applicator (push the D-Squamesapplicator down and then release). The technician removed the samplingdisc with forceps and placed the disc into a pre-labeled 12 well cultureplate. Each subject had two 12 well culture plates for sampling disccollection; one for each leg. Wells 1-6 of each plate were for the sitenearest the knee, while wells 7-12 were used for the site nearest theankle. D-Squames sample plates were placed in shipping boxes with labelscorresponding to the subjects' samples enclosed and placed in a coolerwith dry ice. D-Squames was collected 4 times at the following timepoints, on study days 8, 12, 21, and 29, at approximately 3 hours posttreatment. References: Ertel, K. D., Neumann, P. B., Hartwig, P. M.,Rains, G. Y., and Keswick, B. H., Leg Wash protocol to assess the skinmoisturization potential of personal cleansing products. Int. J. Cosmet.Sci. 21: 383-397 (1999); Fitzpatrick, T. B., The validity andpracticality of sun-reactive skin types I through VI. Arch. Dermatology,124: 869-871 (1988).

Soluble Protein and keratin Analyses: Samples were collected foranalysis using D-Squame Tape Strips. D-Squame tapes were applied on theleg with constant pressure/time, and removed to collect samples of thestratum corneum. Alternative sampling methods using Sebutape and CupScrubs can also be accommodated. Tape strip samples were placed in a 12well plate under frozen conditions (−80° C.) until analysis. Tapesamples were extracted for analysis by placing the tapes inside apolypropylene tube (2 ml) and adding extraction buffer (PBS, pH 7.4,0.04% SDS, Protease Inhibitors) and sonicating for 30 min at 4° C. Thesamples were then centrifuged to remove any insoluble material and thesupernatant is transferred into two deep well plates.

Supernatant samples for keratin analysis were fortified with 2.0% BovineSerum Albumin (BSA) before freezing. The remaining supernatants weretransferred to a second deep well plate for Soluble Protein analysis.Samples were analyzed for Skin analytes (Human Serum Albumin, keratin1,10,11) using validated Millipore™ Multiplex immunoassay methods with aBio-Plex Protein Array Reader system. Soluble protein determinations ofthe supernatants were performed using the Pierce BCA™ Protein assay kitwith the aliquot designated for soluble protein using a validatedmethod. The values obtained for soluble proteins were used to normalizeSkin analyte concentrations were reported as pg/mL or ng/mL and thesoluble proteins were reported as μg/mL. Methods have been validated todemonstrate accuracy, precision, bench top stability, freeze thawstability, short and long term storage stability of the extracts.Extraction efficiency of the methods have been shown to be >70% andreproducible with a single extraction of the tape strips.

Quite surprisingly, the study showed that delivery of a rinse-offcomposition having a lipid to surfactant profile that varied over timeprovided measurable benefits well beyond the stage of high lipiddelivery, as reported below.

Total Protein Results: The results of the total protein from sixconsecutive tape strips as measured by SquameScan 850 were obtained.Referring to FIG. 16, comparative results according to the study areshown. The results showed improved cohesiveness at day 7.0, 14.0, and22.0 measurement points vs. water control.

keratin 1, 10, 11 Results: The results of keratin 1, 10, 11 werenormalized to total soluble protein. Referring to FIG. 17, comparativeresults according to the study are shown. A significant increase ofnormalized keratin 1, 10, 11 as compared to water control at day 7 (100%increase) was observed. The normalized keratin 1, 10, 11 level isfurther increased to 150% vs. water at day 14 and 22. The data isconsistent with literature reports that dry skin dry skin is a conditioncharacterized by hyperproliferation and decreased differentiation. (SeeEngeke, Jensen, Ekanayake-Mudiyanselage and Proksch “Effects of xerosisand aging on epidermal proliferation and differentiation”, Br. J.Dermatology, 137: 219-225 (1997).) This is the first known reportedinstance wherein measurable improvement in total keratin has been shownin the context of use of a rinse-off personal care composition.

Tables 10A-10I show detailed clinical results summary data, and include:Dryness, Corneometer, TEWL, total protein, Ue, Ur, Normalized keratin 1,10, 11, non Normalized keratin 1, 10, 11, and Total Soluble Proteinmeasurements.

TABLE 10A EXPERT DRYNESS GRADES Statistical Grouping Sample (95%Adjusted Standard Attribute Evaluation Size Treatment confidence) MeanError Expert Day 1, 53 [A] Water a 2.794 0.054 Dryness Baseline 53[B]Continuous Lipid a 2.813 0.054 Grades 53 [C] Variable Lipid a 2.7680.054 Expert 24 Hr Post 51 [A] Water b 2.417 0.103 Dryness Wash 6 53[B]Continuous Lipid a 2.062 0.101 Grades (7.0) 51 [C] Variable Lipid a1.972 0.103 Expert 24 Hr Post 48 [A] Water b 2.781 0.103 Dryness Wash 1350 [B]Continuous Lipid a 2.355 0.102 Grades (14.0) 48 [C] Variable Lipida 2.292 0.103 Expert 24 Hr Post 46 [A] Water b 2.804 0.113 Dryness Wash21 49 [B]Continuous Lipid a 2.564 0.111 Grades (22.0) 47 [C] VariableLipid a 2.539 0.113

TABLE 10B CORNEOMETER RESULTS Corneometer Day 1, 53 [A] Water a 20.9360.690 Baseline 53 [B]Continuous Lipid a 20.935 0.690 53 [C] VariableLipid a 20.635 0.690 Corneometer 24 Hr Post 51 [A] Water a 19.084 0.523Wash 6 53 [B]Continuous Lipid b 22.036 0.514 (7.0) 51 [C] Variable Lipidb 22.720 0.523 Corneometer 24 Hr Post 48 [A] Water a 18.102 0.559 Wash13 50 [B]Continuous Lipid b 20.204 0.550 (14.0) 48 [C] Variable Lipid bc20.604 0.558 Corneometer 24 Hr Post 46 [A] Water a 17.513 0.493 Wash 2149 [B]Continuous Lipid b 19.275 0.480 (22.0) 47 [C] Variable Lipid b19.141 0.489

TABLE 10C TEWL Statistical Grouping Sample (95% Adjusted StandardAttribute Evaluation Size Treatment confidence) Mean Error TEWL ReadingsDay 1, 53 [A] Water a 4.324 0.234 (Avg of 2 Probes) Baseline 53[B]Continuous Lipid a 4.484 0.234 53 [C] Variable Lipid a 4.370 0.234TEWL Readings 24 Hr Post 51 [A] Water bc 5.108 0.201 (Avg of 2 Probes)Wash 6 53 [B]Continuous Lipid c 5.217 0.199 (7.0) 51 [C] Variable Lipidabc 4.991 0.201 TEWL Readings 24 Hr Post 48 [A] Water a 6.092 0.190 (Avgof 2 Probes) Wash 13 50 [B]Continuous Lipid a 6.149 0.188 (14.0) 48 [C]Variable Lipid a 5.820 0.190 TEWL Readings 24 Hr Post 46 [A] Water a6.610 0.189 (Avg of 2 Probes) Wash 21 49 [B]Continuous Lipid a 6.3990.186 (22.0) 47 [C] Variable Lipid a 6.301 0.188

TABLE 10D TOTAL PROTEIN (SQUAMESCAN 850 UNIT) Statistical GroupingSample (95% Adjusted Standard Attribute Evaluation Size Treatmentconfidence) Mean Error Sum of First 6 Day 1, 53 [A] Water abc 54.0441.795 Tape Strips Baseline 53 [B]Continuous Lipid ab 53.328 1.795 53 [C]Variable Lipid c 57.722 1.795 Sum of First 6 24 Hr Post 51 [A] Water c60.113 1.770 Tape Strips Wash 6 53 [B]Continuous Lipid ab 50.874 1.74351 [C] Variable Lipid a 50.347 1.784 Sum of First 6 24 Hr Post 48 [A]Water bc 57.452 1.766 Tape Strips Wash 13 50 [B]Continuous Lipid a52.495 1.739 48 [C] Variable Lipid a 50.203 1.780 Sum of First 6 24 HrPost 46 [A] Water c 56.235 1.864 Tape Strips Wash 21 49 [B]ContinuousLipid ab 52.024 1.828 47 [C] Variable Lipid a 49.193 1.864

TABLE 10E ELASTIC DEFORMATION (CUTOMETER RESULTS) Statistical GroupingSample (95% Adjusted Standard Attribute Evaluation Size Treatmentconfidence) Mean Error Ue—Elastic Day 1, 53 [A] Water a 0.710 0.033Deformation of Baseline 53 [B]Continuous Lipid a 0.704 0.033 Skin 53 [C]Variable Lipid a 0.669 0.033 Ue—Elastic 1 Hr Post 48 [A] Water a 0.4180.016 Deformation of Wash 14 49 [B]Continuous Lipid b 0.458 0.016 Skin(14.1) 48 [C] Variable Lipid b 0.472 0.016 Ue—Elastic 1 Hr Post 46 [A]Water a 0.390 0.016 Deformation of Wash 21 48 [B]Continuous Lipid b0.432 0.016 Skin (21.1) 47 [C] Variable Lipid b 0.421 0.016

TABLE 10F ELASTIC RECOVERY (CORNEOMETER RESULTS) Statistical GroupingSample (95% Adjusted Standard Attribute Evaluation Size Treatmentconfidence) Mean Error Ur—Elastic Day 1, 53 [A] Water a 0.665 0.029Deformation Baseline 53 [B]Continuous Lipid a 0.647 0.029 Recovery 53[C] Variable Lipid a 0.630 0.029 Ur—Elastic 1 Hr Post 50 [A] Water a0.432 0.020 Deformation Wash 7 52 [B]Continuous Lipid b 0.470 0.019Recovery (7.1) 50 [C] Variable Lipid b 0.500 0.020 Ur—Elastic 1 Hr Post48 [A] Water a 0.401 0.018 Deformation Wash 14 49 [B]Continuous Lipid b0.446 0.018 Recovery (14.1) 48 [C] Variable Lipid b 0.466 0.018Ur—Elastic 1 Hr Post 46 [A] Water a 0.378 0.018 Deformation Wash 21 48[B]Continuous Lipid b 0.441 0.018 Recovery (21.1) 47 [C] Variable Lipidb 0.435 0.018

TABLE 10G LOG(NORMALIZED KERATIN 1, 10, 11 TO SOLUBLE PROTEIN IN ng/ug)Statistical Grouping Sample (95% Adjusted Standard Attribute EvaluationSize Treatment confidence) Mean Error Log(keratin Day 1, 51 [A] Water a1.655 0.068 1, 10, 11 Baseline 53 [B]Continuous Lipid a 1.657 0.067Ratio[Soluble 51 [C] Variable Lipid a 1.642 0.068 Protein]) Log(keratin24 Hr Post 51 [A] Water a 1.730 0.072 1, 10, 11 Wash 6 53 [B]ContinuousLipid b 2.040 0.071 Ratio[Soluble (7.0) 51 [C] Variable Lipid b 2.0370.072 Protein]) Log(keratin 24 Hr Post 48 [A] Water a 1.633 0.076 1, 10,11 Wash 13 50 [B]Continuous Lipid bc 1.942 0.074 Ratio[Soluble (14.0) 48[C] Variable Lipid c 2.071 0.076 Protein]) Log(keratin 24 Hr Post 46 [A]Water a 1.341 0.082 1, 10, 11 Wash 21 49 [B]Continuous Lipid b 1.6780.080 Ratio[Soluble (22.0) 47 [C] Variable Lipid b 1.771 0.081 Protein])

TABLE 10H Log (Non Normalized keratin 1, 10, 11 in ng/ml) StatisticalGrouping Sample (95% Adjusted Standard Attribute Evaluation SizeTreatment confidence) Mean Error Log(keratin Day 1, 51 [A] Water ab3.806 0.057 1, 10,11 Baseline 53 [B]Continuous Lipid a 3.741 0.057 NoNormalization 51 [C] Variable Lipid ab 3.794 0.057 Log(keratin 24 HrPost 51 [A] Water b 3.704 0.041 1, 10, 11 Wash 6 53 [B]Continuous Lipidab 3.645 0.040 No Normalization (7.0) 51 [C] Variable Lipid a 3.5760.041 Log(keratin 24 Hr Post 48 [A] Water b 3.750 0.057 1, 10, 11 Wash13 50 [B]Continuous Lipid ab 3.617 0.056 No Normalization (14.0) 48 [C]Variable Lipid a 3.606 0.057 Log(keratin 24 Hr Post 46 [A] Water c 3.7850.054 1, 10, 11 Wash 21 49 [B]Continuous Lipid bc 3.726 0.052 NoNormalization (22.0) 47 [C] Variable Lipid ab 3.644 0.053

TABLE 10I LOG(SOLUBLE PROTEIN IN ug/ml) Statistical Grouping Sample (95%Adjusted Standard Attribute Evaluation Size Treatment confidence) MeanError Log(Soluble Day 1, 51 [A] Water a 2.150 0.072 Protein) Baseline 53[B]Continuous Lipid a 2.085 0.071 51 [C] Variable Lipid a 2.155 0.072Log(Soluble 24 Hr Post 51 [A] Water b 1.971 0.081 Protein) Wash 6 53[B]Continuous Lipid a 1.605 0.080 (7.0) 51 [C] Variable Lipid a 1.5420.081 Log(Soluble 24 Hr Post 48 [A] Water b 2.119 0.084 Protein) Wash 1350 [B]Continuous Lipid a 1.672 0.083 (14.0) 48 [C] Variable Lipid a1.535 0.084 Log(Soluble 24 Hr Post 46 [A] Water c 2.446 0.089 Protein)Wash 21 49 [B]Continuous Lipid b 2.040 0.088 (22.0) 47 [C] VariableLipid ab 1.884 0.089

Based on the results as reported above, further analysis of the resultswas undertaken to determine whether the measured skin health correlateswith the biomarker measurements as obtained in the study. The resultsare reported in FIGS. 18 through 21. Referring to FIG. 18 and FIG. 19,it is evident that the combined biomarkers keratin 1, 10, 11 are keyindicators for skin dryness and corneometer hydration, and the measuredincreases in these biomarkers trend well with objective measures ofimprovement in skin dryness and hydration. Referring again to FIG. 19and FIG. 20, the analysis shows that total NMFs are key indicators forTEWL and elastic recovery (Ur), and the measured increases in thesebiomarkers trend well with objective measures of improvement retentionof moisture and elasticity. More generally, as shown in FIG. 18 and FIG.21, the inflammatory cytokines, NMFs, keratins and total proteinmeasured increases all are predictive of physical improvements.

These results are the first known report of measurement of biomarkersfor demonstrating improvement of skin as shown by objective measures. Wehave demonstrated that use of the biomarker panels as described hereinsupports the formulation of personal care products, particularly in therinse-off context, that achieve objective improvement of skin health. Inaddition, the methods described herein enable the identification andhence the formulation of products that can deliver sustained benefitsbeyond the treatment period.

Example 3: Calculation of Improvement Indices for Physical Measurementsand Biomarkers

The above described results indicate that the variable lipid body washdelivers significant improvements in several standardmoisturization/tissue health measures (dryness grades, corneometerhydration, and TEWL). We believe we are the first to report in therinse-off context, the results show significant improvement in skinelasticity as compared to the water treatment control. The total proteinresults further reveal significant improvement in stratum corneumcohesiveness. Taken together, these findings support the conclusion thatthe petrolatum depositing body wash wherein hydrophobic benefit phase isdelivered in varying amounts over a treatment cycle according variousembodiments as described herein improves the overall condition of skin.Improvement indices for each of the measures described herein above areshown in Table 11A and B.

TABLE 11A Inventive Example A vs. p value Improvement Index WaterControl (base size n = 50) a) Skin Elastic Extension (Ue) 16 p = 0.003Improvement Index b) Skin Elastic Recovery (Ur) 21 p = 0.0004Improvement Index c) Skin Elasticity (R7) 4 p = 0.05 Improvement Indexd) Stratum Corneum Cohesiveness 23 p < 0.0001 Improvement Index e)keratin Improvement Index 172 p < 0.0001 f) Visual Dryness Improvement1.5 p < 0.0001 (Dryness Reduction at 3 hours after three weeks ofproduct treatment) g) Corneometer Improvement 3.6 p < 0.0001 (Increasein Corneometer at 3 hours after three weeks of product treatment) h)TEWL Improvement (TEWL 0.5 P = 0.016 reduction at 3 hours after threeweeks of product treatment)

TABLE 11B Inventive Example A vs. p value Improvement Index WaterControl (base size n = 50) a) Inflammatory Cytokines 113 P < 0.0001 b)NMFs 5.7 p = 0.0737 c) Ceramides 57 p = 0.0008 d) Fatty Acids 43 p =0.0015 e) Total protein 8.2 P = 0.0002Calculation Improvement Index

-   -   1) Calculation of Skin Elasticity Improvement Index        -   a) Elastic Extension (U_(e)) Improvement Index is calculated            as:            [(U _(e))^(P) _(end)−(U _(e))^(c) _(end)]/(U _(e))^(c)            _(end)*100−[(U _(e))^(P) _(ini)−(U _(e))^(c) _(ini)]/(U            _(e))^(c) _(ini)*100 wherein            -   (U_(e))^(c) _(ini) is the initial elastic extension                parameter at the beginning of the water control leg;            -   (U_(e))^(P) _(ini) is the initial elastic extension                parameter at the beginning of the test product leg;            -   (U_(e))^(c) _(end) is the final elastic extension                parameter at the end of the water control leg;                -   (U_(e))^(P) _(end) is the final elastic extension                    parameter at the end of the test product leg.        -   b) Elastic Recovery (U_(r)) Improvement Index is calculated            as:            [(U _(r))^(P) _(end)−(U _(r))^(c) _(end)]/(U _(r))^(c)            _(end)*100−[(U _(r))^(P) ini−(U _(r))^(c) ini]/(U _(r))^(c)            _(ini)*100 wherein            -   (U_(r))^(c) _(ini) is the initial elastic recovery                parameter at the beginning of the water control leg;            -   (U_(r))^(P) _(ini) is the initial elastic recovery at                the beginning of the test product leg;            -   (U_(r))^(c) _(end) is the final elastic recovery at the                end of the water control leg;                -   (U_(r))^(P) _(end) is the final elastic recovery at                    the end of the test product leg.        -   c) Elasticity (R₇) Improvement Index is calculated as:            [(R ₇)^(P) _(end)−(R ₇)^(c) _(end)]/(R ₇)^(c) _(end)*100−[(R            ₇)^(P) _(ini)−(R ₇)^(c) _(ini)]/(R ₇)^(c) _(ini)*100 wherein            -   (R₇)^(c) _(ini) is the initial elasticity at the                beginning of the water control leg;            -   (R₇)^(P) _(ini) is the initial elasticity at the                beginning of the test product leg;            -   (R₇)^(c) _(end) is the final elasticity at the end of                the water control leg;                -   (R₇)^(P) _(end) is the final elasticity at the end                    of the test product leg.    -   2) Calculation of Stratum Corneum Cohesiveness Improvement Index        -   Stratum Corneum Cohesiveness Improvement Index is calculated            as:            [(Protein)^(C) _(end)−(Protein)^(P) _(end)]/(Protein)^(C)            _(end)*100−[(Protein)^(C) _(ini)−(Protein)^(P)            _(ini)]/(Protein)^(C) _(ini)*100 wherein        -   (Protein)^(c) _(ini) is the sum of initial protein            absorption of tape 1 to tape 6 at the beginning of the water            control leg;        -   (Protein_(e))^(P) _(ini) is the sum of initial protein            absorption of tape 1 to tape 6 at the beginning of the test            product leg;        -   (Protein)^(c) _(end) is the sum of final protein absorption            of tape 1 to tape 6 at the end of the water control leg;        -   (Protein)^(P) _(end) is the sum of final protein absorption            of tape 1 to tape 6 at the end of the test product leg.    -   3) Calculation of Keratin 1, 10, 11 Improvement Index        -   Keratin 1, 10, 11 Improvement Index is calculated as:            [(keratin)^(P) _(end)−(keratin)^(C) _(end)]/(keratin)^(C)            _(end)*100−[(keratin)^(P) _(ini)−(keratin)^(C)            _(ini)]/(keratin)^(C) _(ini)*100 wherein        -   (keratin)^(c) _(ini) is the initial keratin 1, 10, 11            normalized to total soluble protein at the beginning of the            water control leg;        -   (keratin)^(P) _(ini) is the initial keratin 1, 10, 11            normalized to total soluble protein at the beginning of the            test product leg;        -   (keratin)^(c) _(end) is the final keratin 1, 10, 11            normalized to total soluble protein at the end of the water            control leg;        -   (keratin)^(P) _(end) is the final keratin 1, 10, 11            normalized to total soluble protein at end of the test            product leg.    -   4) Calculation of Inflammatory Cytokine Improvement Index        -   Cytokine Improvement Index is calculated as:            [(Cytokine)^(c) _(end)−(Cytokine)P_(end)]/(Cytokine)^(C)            _(end)*100−[(Cytokine)^(c) _(ini)−(Cytokine)^(P)            _(ini)]/(Cytokine)^(C) _(ini)*100 wherein        -   (Cytokine)^(c) _(ini) is the initial Cytokine normalized to            total soluble protein at the beginning of the water control            leg;        -   (Cytokine)^(P) _(ini) is the initial Cytokine normalized to            total soluble protein at the beginning of the test product            leg;        -   (Cytokine)^(c) _(end) is the final Cytokine normalized to            total soluble protein at the end of the water control leg;        -   (Cytokine)^(P) _(end) is the final Cytokine normalized to            total soluble protein at end of the test product leg.    -   5) Calculation of NMF Improvement Index        -   NMF Improvement Index is calculated as:            [(NMF)^(P) _(end)−(NMF)^(c) _(end)]/(NMF)^(C)            _(end)*100−[(NMF)^(P) _(ini)−(NMF)^(c) _(ini)]/(NMF)^(C)            _(ini)*100 wherein        -   (NMF)^(c) _(ini) is the initial NMF normalized to total            soluble protein at the beginning of the water control leg;        -   (NMF)^(P) _(ini) is the initial NMF normalized to total            soluble protein at the beginning of the test product leg;        -   (NMF)^(c) _(end) is the final NMF normalized to total            soluble protein at the end of the water control leg;        -   (NMF)^(P) _(end) is the final NMF normalized to total            soluble protein at end of the test product leg.    -   6) Calculation of Ceramides Improvement Index        -   Ceramides Improvement Index is calculated as:            [(Ceramides)^(P) _(end)−(Ceramides)^(c)            _(end)]/(Ceramides)^(C) _(end)*100−[(Ceramides)^(P)            _(ini)−(Ceramides)^(c) _(ini)]/(Ceramides)^(C) _(ini)*100            wherein        -   (Ceramides)^(c) _(ini) is the initial Ceramides normalized            to total soluble protein at the beginning of the water            control leg;        -   (Ceramides)^(P) _(ini) is the initial Ceramides normalized            to total soluble protein at the beginning of the test            product leg;        -   (Ceramides)^(c) _(end) is the final Ceramides normalized to            total soluble protein at the end of the water control leg;        -   (Ceramides)^(P) _(end) is the final Ceramides normalized to            total soluble protein at end of the test product leg.    -   7) Calculation of Fatty Acids Improvement Index        -   Fatty Acids Improvement Index is calculated as:            [(Fatty Acids)^(p) _(end)−(Fatty Acids)^(c) _(end)]/(Fatty            Acids)^(C) _(end)*100−[(Fatty Acids)P_(ini)−(Fatty            Acids)^(c) _(ini)]/(Fatty Acids)^(C) _(ini)*100 wherein        -   (Fatty Acids)^(c) _(ini) is the initial Fatty Acids            normalized to total soluble protein at the beginning of the            water control leg;        -   (Fatty Acids)^(P) _(ini) is the initial Fatty Acids            normalized to total soluble protein at the beginning of the            test product leg;        -   (Fatty Acids)^(c) _(end) is the final Fatty Acids normalized            to total soluble protein at the end of the water control            leg;        -   (Fatty Acids)^(P) _(end) is the final Fatty Acids normalized            to total soluble protein at end of the test product leg.    -   8) Calculation of Total Protein Improvement Index        -   Cytokine Improvement Index is calculated as:            [(Total Protein)^(c) _(end)−(Total Protein)^(p)            _(end)]/(Total Protein)^(C) _(end)*100−[(Total Protein)^(c)            _(ini)−(Total Protein)^(p) _(ini)]/(Total Protein)^(C)            _(ini)*100 wherein        -   (Total Protein)^(c) _(ini) is the initial Total Protein at            the beginning of the water control leg;        -   (Total Protein)^(P) _(ini) is the initial Total Protein at            the beginning of the test product leg;        -   (Total Protein)^(c) _(end) is the final Total Protein at the            end of the water control leg;        -   (Total Protein)^(P) _(end) is the final Total Protein at end            of the test product leg.

Example 4: Biomarker Assay Methods

Cytokine, SkinMAP and Soluble Protein: Samples are collected forBiomarker analysis using D-Squame Tape Strips. D-Squame tapes areapplied on the site of interest (Scalp, Leg, Face, Underarm, Forearm)with constant pressure/time, and removed to collect samples of thestratum corneum. Alternative sampling methods using Sebutape and CupScrubs can also be accommodated. Tape strip samples are placed in a 12well plate under frozen conditions (−80° C.) until analysis. Tapesamples are extracted for analysis by placing the tapes inside apolypropylene tube (2 ml) and adding extraction buffer (PBS, pH 7.4,0.04% SDS, Protease Inhibitors) and sonicating for 30 min at 4° C. Thesamples are then centrifuged to remove any insoluble material and thesupernatant is transferred into two deep well plates. Supernatantsamples for Cytokine/SkinMAP analysis are fortified with 2.0% BovineSerum Albumin (BSA) before freezing. The remaining supernatants aretransferred to a second deep well plate for Soluble Protein analysis.Samples are analyzed for Cytokines (IL-1α, IL-1ra, IL-8) and Skinanalytes (Human Serum Albumin, keratin 1,10,11 and Involucrin) usingvalidated Millipore™ Multiplex immunoassay methods with a Bio-PlexProtein Array Reader system. Soluble protein determinations of thesupernatants are performed using the Pierce BCA™ Protein assay kit withthe aliquot designated for soluble protein using a validated method. Thevalues obtained for soluble proteins are used to normalize the Cytokineand Skin Map data. Cytokine and Skin analyte concentrations are reportedas pg/mL or ng/mL and the soluble proteins are reported as μg/mL.Methods have been validated to demonstrate accuracy, precision, benchtop stability, freeze thaw stability, short and long term storagestability of the extracts. Extraction efficiency of the methods havebeen shown to be >70% and reproducible with a single extraction of thetape strips.

Lipids: Samples for lipid analysis are collected using D-Squame tapessimilar to protein biomarker analysis. The samples are extracted using asolution (400 mM Urea and 2.0% SDS) with tapes suspended in thissolution in scintillation vials. The tapes are sonicated and the extractsuspension is removed to a separate scintillation vial for lipidextraction. Chloroform/Methanol is added according to the method withstable label internal standards for lipid extraction. The organic layercontaining the lipids are removed and dried for analysis of lipids bySFC-HPLC/MS/MS. Total protein (soluble and insoluble) in the tapeextracts were determined using BCA™ (Pierce) method.

Example 5: Dryness Grading Procedure

The skin on the subject's lower leg will be graded for dryness by aqualified grader according to the scales below. Each subject's lowerlegs will be visually evaluated at baseline on Study Day 8 (prior toproduct application), as a prerequisite for continuation into thetreatment phase. Only those subjects with baseline dryness scores ≧2.5and <4.0 on the scale below on each of the defined sites will qualifyfor enrollment into the treatment phase of the study. Each site on thesubject's legs will be graded 9 more times: 3 hours post 1^(st), 3^(rd),5^(th) 14^(th) and 21^(st) treatments; 24 hours post 4^(th), 13^(th) and21^(st), treatments, 48 hours post 21^(st) treatment. Subjects willacclimate for a minimum of thirty minutes in an environmentallycontrolled room (maintained at 70° F.±2 and 30-45% relative humidity)prior any visual evaluations of their legs. The same skin grader will beused for the duration of the study. Data will be recorded electronicallyusing the Sponsor's direct data entry and data capture programs.

If a termination score is suspected, the Investigator will arrange tohave the subject's legs evaluated by a qualified Grader between threeand four hours (if possible) after the previous treatment. If necessary,the subject will be asked to return to the test facility at a later timefor this visual evaluation. If the Grader determines that the subjecthas attained dryness grade ≧5.0 on any site the subject will be droppedand a Subject prop Form (DCF DROP) will be completed. The scoresassigned to the sites at the time of the drop will be considered thefinal evaluation scores. Final instrumental measurements will also bemade at this time. These visual and instrumental results will beincluded in the study data set. If the Investigator deems that theirritation does not interfere with the adjacent sites and there is noother reason for withdrawal, the subject's participation in the studywill not be discontinued and treatment and evaluations of the remainingsites will be continued until the end of the study.

Materials: Visual evaluations will be done with the aid of a LuxoIlluminated Magnifying Lamp (Model KFM-1A) which provides 2.75×magnification and which has a shadow-free circular fluorescent lightsource (General Electric Cool White, 22 watt 8″ Circline bulb).

Grade^(a) Dryness^(b) 0.0 perfect skin 1.0 patches of checking and/orslight powderiness, occasional patches of small scales may be seen,distribution generalized 2.0 generalized slight powderiness, earlycracking or occasional small lifting scales may be present 3.0generalized moderate powderiness and/or moderate cracking and scales 4.0generalized heavy powderiness and/or heavy cracking and lifting scales5.0 generalized high cracking and lifting scales, eczematous change maybe present but not prominent, may see bleeding cracks 6.0 generalizedsevere cracking, bleeding cracks and eczematous changes may be present,large scales may be sloughing off ^(a)half-unit grades may be used ifnecessary ^(b)‘generalized’ refers to situations where more than 50% ofthe application area is affected

Example 6: Corneometer Measurements

Measurement Procedure: Skin hydration based upon the measurement ofcapacitance will be assessed using the Corneometer® 825. Thesenon-invasive measurements will be taken in duplicate on each site of thesubjects' legs a total of 10 times: at baseline, prior to 1^(st)treatment; 3 hours post 1^(st), 3^(rd), 5^(th) 14^(th) and 21^(st)treatments; 24 hours post 4^(th), 13^(th) and 21^(st), treatments, 48hours post 21^(st) treatment after the visual assessment have beencompleted. Subjects will acclimate for a minimum of thirty minutes in anenvironmentally controlled room (maintained at 70° F.±2 and 30-45%relative humidity) prior to non-invasive instrumental measurements takenon their legs. Data will be recorded electronically using the Sponsor'sdirect data entry and data capture programs.

Operating Instructions: The taking of these measurements will be doneaccording to the test facility's SOP's and/or the Sponsors InstrumentOperation Manual.

-   -   1) The Corneometer values are arbitrary units for electrical        impedance. At baseline these values typically fall within a        similar range. If a measurement is outside of this observed        range re-take the measurement following the procedure in the        Sponsor's Instrument Operational Manual. If the value continues        to be outside of the observed range then the study coordinator        will contact the Clinical Trial Manager to determine if further        action is needed. The values along with the subject # and visit        are to be recorded on an error log sheet. Post-treatment        measurements will also typically result in values within a        similar range depending on the treatment. Post-treatment        measurements outside this range will also be re-checked and        recorded as above.    -   2) The instrument will be operated by only trained operators.        Training record must be on file and up to date. The same        instrument(s) and operator(s) will be used throughout the study.    -   3) Only use Kimwipes to wipe the end of the probe. The probe        should be wiped with a Kimwipe between each measurement.    -   4) At the end of the evaluation session back up the data        collected for that period following the instructions in the        Sponsors Instrument Operation Manual. In addition you will print        a hard copy of the data collected for the period.

Example 7: TEWL Measurements

Measurement Procedure: The integrity of the stratum corneum barrier willbe assessed by transepidermal water loss using the DermaLab®Evaporimeter equipped with dual probes. Single non-invasive measurementswill be taken on each site of the subjects' lower leg a total of 9times: at baseline, prior to 1^(st) treatment; 3 hours post 3^(rd),5^(th) 14^(th) and 21^(st) treatments; 24 hours post 4^(th), 13^(th) and21^(st), treatments, 48 hours post 21^(st) treatment after the visualassessment and instrumental measurements have been completed. Due to thenumber of subjects and the time involved two instruments will be usedduring the study. Subjects will be assigned to one of the twoinstruments on the basis of their assigned subject number for theduration of the study. Each measurement consists of readings collectedfor 60 seconds with the mean of the last 20 seconds recorded from bothprobes (Channel A and Channel B). Subjects will acclimate for a minimumof thirty minutes in an environmentally controlled room (maintained at70° F.±2 and 30-45% relative humidity) prior to instrumentalmeasurements. Data will be recorded electronically using the Sponsor'sdata capture programs.

Operating Instructions: The taking of these measurements will be doneaccording to the test facility's SOP's and/or the Sponsors InstrumentOperation Manual.

-   -   1) The DermaLab values are arbitrary units of transepidermal        water loss from the skin. At baseline these values typically        fall within a similar range. If a measurement is outside of this        observed range re-take the measurement following the procedure        in the Sponsor's Instrument Operational Manual. If the value        continues to be outside of the observed range then the study        coordinator will contact the Clinical Trial Manager to determine        if further action is needed. The values along with the subject #        and visit are to be recorded on an error log sheet.        Post-treatment measurements will also typically result in values        within a similar range depending on the treatment.        Post-treatment measurements outside this range will also be        re-checked and recorded as above.    -   2) If one of the two probes fails during any measurement phase        all subsequent measurements will be taken with the remaining        single probe.    -   3) The instrument(s) will be operated by only trained operators.        Training record must be on file and up to date. The same        instrument(s) and operator(s) will be used throughout the study.    -   4) Only use Kimwipes to wipe the end of the probe. The probe        should be wiped with a Kimwipe between each measurement.    -   5) At the end of the evaluation session back up the data        collected for that period following the instructions in the        Sponsors Instrument Operation Manual. In addition you will print        a hard copy of the data collected for the period.

All percentages, parts and ratios are based upon the total weight of thecompositions used in accordance with the present disclosure, unlessotherwise specified. All such weights as they pertain to listedingredients are based on the active level and, therefore; do not includesolvents or by-products that may be included in commercially availablematerials, unless otherwise specified. The term “weight percent” may bedenoted as “wt. %” herein. Except where specific examples of actualmeasured values are presented, numerical values referred to hereinshould be considered to be qualified by the word “about.”

All molecular weights as used herein are weight average molecularweights expressed as grams/mole, unless otherwise specified.

The dimensions and values disclosed herein are not to be understood asbeing strictly limited to the exact numerical values recited. Instead,unless otherwise specified, each such dimension is intended to mean boththe recited value and a functionally equivalent range surrounding thatvalue. For example, a dimension disclosed as “40 mm” is intended to mean“about 40 mm.”

All documents cited in the Detailed Description are, in relevant part,incorporated herein by reference; the citation of any document is not tobe construed as an admission that it is prior art with respect to thepresent disclosure. To the extent that any meaning or definition of aterm in this document conflicts with any meaning or definition of thesame term in a document incorporated by reference, the meaning ordefinition assigned to that term in this document shall govern.

While particular embodiments as disclosed herein have been illustratedand described, it would be obvious to those skilled in the art thatvarious other changes and modifications can be made without departingfrom the spirit and scope of the disclosure. It is therefore intended tocover in the appended claims all such changes and modifications that arewithin the scope of this disclosure.

What is claimed is:
 1. A screening method for identifying a body washcomposition as effective at improving the health of human skin,comprising: a. during a treatment period comprising at least onetreatment, contacting a skin surface of a human subject with a body washcomposition during a treatment period, wherein the body wash compositionis washed off after each application; b. at least once during thetreatment period extracting from the epidermis of the human subject (i)at least one biomarker selected from the group consisting of IL1 rα andIL 1α, (ii) at least one biomarker selected from the group consisting ofTrans-Urocanic Acid, Citrulline, Glycine, Histidine, Ornithine, Proline,2 Pyrrolidone 5 acid, and Serine,(iii) at least one biomarker that is aceramide, (iv) at least one biomarker that is a fatty acid, and (v)total protein; c. measuring an amount of each biomarker extracted; andd. identifying the body wash composition as effective if the amount ofeach biomarker is shifted in a direction of improved skin health withtotal protein decreasing.
 2. The method according to claim 1, whereinthe treatment period includes two or more treatments.
 3. The methodaccording to any one of claims 1 and 2, wherein the biomarkers areextracted and measured before a first treatment of the treatment periodand after the first treatment.
 4. The method according to claim 3,wherein the body wash composition remains on the skin surface forbetween about 0.5 minutes and about 50 minutes prior to the washing offof the body wash composition.
 5. The method according to claim 4,wherein the treatment period includes at least two treatments, andwherein the body wash composition comprises a lipid component and asurfactant component that are present in a first ratio at the start ofthe treatment period and are present in a different ratio at the end ofthe treatment period.
 6. The method according to claim 5, wherein theratio of lipid to surfactant increases during the treatment period.